The Sequence Listing submitted Oct. 20, 2023 as an xml file named “37759_0397_U3_Sequence_Listing,” created on Oct. 19, 2023, and having a size of 15,871 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).
Vision loss associated with ischemic diseases, such as retinopathy of prematurity, diabetic retinopathy and optic neuropathy, are often due to the growth of malfunctional retinal capillaries, in response to retinal ischemia. Significant progress has been made in combating abnormal vascular proliferation, including laser photocoagulation, anti-angiogenic vascular endothelial growth factor (VEGF) inhibitors and angiostatic steroids. Nevertheless, these therapeutic approaches fail to treat ischemic areas with a large degree of tissue injury in addition to local and systemic complications associated with their use. Thus, there is an urgent need to develop new therapeutic alternatives to combat early retinal ischemia and thus diminish subsequent destructive neovascularization.
The p75NTR, a common receptor for all neurotrophins along with their precursor forms, can exert multiple functions, including cell survival, death or angiogenesis, according to cell context. Among increasing prospective stem cells markers, the p75NTR receptor, also known as CD271, enriches several progenitor/stem cell subtypes. P75NTR was first identified as a genuine neural crest stem cell marker. Since then, it has been widely used to isolate putative stem cells from neural crest-derived tissues. The p75NTR was shown to directly inhibit the differentiation of MSCS into multiple cell types. Several studies reported a causal relationship of p75NTR in mediating retinal inflammation, barrier dysfunction and development of acellular capillaries. Nonetheless, p75NTR expression and function in MSCs biology, as well as its underlying mechanisms, have not been fully studied. In the current study, the impact of modulating p75NTR expression or activity on the surface of mesenchymal stem cells (MSCs) in increasing their vascular homing and repair in ischemic retina using an ischemia/reperfusion (I/R) mouse model was evaluated. Further, the protective action of pharmacological inhibition of p75NTR on improving visual acuity post I/R injury was investigated.
Disclosed are methods of treating an ocular disease associated with retinal ischemic injury in a subject comprising administering to the subject in need thereof a p75NTR modulator.
Disclosed are methods of treating an ocular disease associated with retinal ischemic injury in a subject comprising administering to the subject in need thereof a mesenchymal stem cell (MSC) comprising decreased p75NTR expression or activity.
Disclosed are methods of treating an ocular disease associated with retinal ischemic injury in a subject comprising administering to the subject in need thereof a mesenchymal stem cell (MSC) secretome collected from a MSC comprising decreased p75NTR expression or activity.
Disclosed are methods of improving visual acuity in a subject having a retinal ischemic injury comprising administering to the subject in need thereof a p75NTR modulator.
Disclosed are methods of improving visual acuity in a subject having a retinal ischemic injury comprising administering to the subject in need thereof a mesenchymal stem cell (MSC) comprising decreased p75NTR expression or activity.
Disclosed are methods of improving visual acuity in a subject having a retinal ischemic injury comprising administering to the subject a mesenchymal stem cell (MSC) secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
Disclosed are methods of preventing ischemia-induced retinal capillary degeneration in a subject comprising administering to the subject in need thereof a p75NTR modulator, wherein the p75NTR modulator inhibits the expression or activity of p75NTR in one or more MSCs in the retina of the subject.
Disclosed are methods of preventing ischemia-induced retinal capillary degeneration in a subject comprising administering to the subject one or more MSCs comprising decreased p75NTR expression or activity.
Disclosed are methods preventing ischemia-induced retinal capillary degeneration in a subject comprising administering to the subject a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
Disclosed are methods of increasing vascular protection in an ischemic retina of a subject comprising administering to the retina of the subject in need thereof a p75NTR modulator, wherein the p75NTR modulator inhibits expression or activity of p75NTR in one or more MSCs in the retina of the subject.
Disclosed are methods of increasing vascular protection in an ischemic retina of a subject comprising administering to the retina of the subject one or more MSCs comprising decreased p75NTR expression or activity.
Disclosed are methods of increasing vascular protection in an ischemic retina of a subject comprising administering to the retina of the subject a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
Disclosed are methods of increasing vascular homing of one or more MSCs at a site of ischemic injury in a subject comprising administering to the subject in need thereof a p75NTR modulator, wherein the p75NTR modulator inhibits expression or activity of p75NTR in the one or more MSCs.
Disclosed are methods of increasing vascular homing of one or more MSCs at a site of ischemic injury in a subject comprising administering to the subject one or more MSCs comprising decreased p75NTR expression or activity.
Disclosed are methods of increasing vascular homing of one or more MSCs at a site of ischemic injury in a subject comprising administering to the subject a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
Disclosed are methods of altering a MSC secretome comprising inhibiting the expression or activity of p75NTR in the MSC. In some aspects, altering a MSC secretome can be improving a MSC's secretome.
Disclosed are methods of increasing survival factors in a HRE comprising contacting the HRE with one or more MSCs having decreased p75NTR expression or activity.
Disclosed are methods of increasing angiogenic effects of a HRE comprising contacting the HRE with one or more MSCs having decreased p75NTR expression or activity, wherein HRE migration is increased.
Disclosed are methods of decreasing urinary albumin excretion rate in a subject having diabetes comprising administering to the subject a p75NTR modulator.
Disclosed are methods of decreasing inflammation in the kidney of a subject having diabetes comprising administering to the subject a p75NTR modulator.
Disclosed are methods of decreasing IL-1β in a subject having diabetes comprising administering to the subject a p75NTR modulator, wherein IL-1β is decreased in one or both kidneys after administration of the p75NTR modulator.
Disclosed are methods of decreasing IL-6 in a subject having diabetes comprising administering to the subject in need thereof a p75NTR modulator, wherein IL-6 is decreased in one or both kidneys after administration of the p75NTR modulator.
Disclosed are methods of decreasing TGF-β in a subject having diabetes comprising administering to the subject in need thereof a p75NTR modulator, wherein TGF-β is decreased in one or both kidneys after administration of the p75NTR modulator.
Disclosed are methods of decreasing one or more markers of fibrosis in a subject having diabetes comprising administering to the subject in need thereof a p75NTR modulator, wherein one or more markers of fibrosis is decreased in one or both kidneys after administration of the p75NTR modulator.
Disclosed are methods of decreasing fibrotic tissue in a kidney of a subject having diabetes comprising administering to the subject in need thereof a p75NTR modulator.
Disclosed are methods of treating late stage diabetes in a subject comprising administering to the subject in need thereof a p75NTR modulator.
Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.
The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.
It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms “a ”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a MSC” includes a plurality of such MSCs, reference to “the p75NTR modulator” is a reference to one or more p75NTR modulators and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “subject” or “patient” can be used interchangeably and refer to any organism to which a composition, a p75NTR modulator, a MSC, or a MSC secretome of this invention may be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as non-human primates, and humans; avians; domestic household or farm animals such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats and guinea pigs; rabbits; fish; reptiles; zoo and wild animals). Typically, “subjects” are animals, including mammals such as humans and primates; and the like.
By “treat” is meant to administer a protein, nucleic acid, or composition of the invention to a subject, such as a human or other mammal (for example, an animal model), that has an increased susceptibility for developing retinal ischemia and/or diabetes or that has retinal ischemia and/or diabetes, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease or condition.
By “prevent” is meant to minimize the chance that a subject who has an increased susceptibility for developing retinal ischemia and/or diabetes actually develops the disease/condition or otherwise develops a cause of symptom thereof.
As used herein, the terms “administering” and “administration” refer to any method of providing a disclosed compositions, a p75NTR modulator, a MSC, or a MSC secretome to a subject. Such methods are well known to those skilled in the art and include, but are not limited to: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition. In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, or an efficacious route of administration for a disclosed composition or a disclosed protein so as to treat a subject or induce an immune response. In an aspect, the skilled person can also alter or modify an aspect of an administering step so as to improve efficacy of a disclosed protein, nucleic acid, composition, or a pharmaceutical preparation.
As used throughout, “po” means oral administration. For example, interventional treatment with LM11A-31 po refers to oral administration of LM11A-31.
“Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.
Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.
In some aspects, a p75NTR modulator can be a compound, nucleic acid sequence, or peptide.
In some aspects, the p75NTR modulator is LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be, but is not limited to, LM11A-24, THX-B, EVT901 or any derivatives thereof. In some aspects, the p75NTR modulator is a mimetic of a neurotrophin. For example, any of the p75NTR modulators described in U.S. Pat. No. 8,916,556, incorporated by reference in its entirety herein, can be used in the disclosed methods.
In some aspects, the p75NTR modulator can be any compound, nucleic acid sequence, or peptide that reduces p75NTR mRNA expression. For example, in some aspects, the p75NTR modulator can be siRNA or antisense RNA. In some aspects, siRNA can be delivered using an expression vector. An example of a p75NTR-specific siRNA expression vector can be constructed by ligating a double-stranded hairpin oligonucleotide: 5′-GAT CCG AGG ATC GGA GGC TTG TCA TTC AAG AGA TGA CAA GCC TCC GAT CCT CTT TTT TGG AAA-3′ (SEQ ID NO:1), containing a p75NTR-specific siRNA sequence (underlined), into a vector. In some aspects, the p75NTR siRNA is a known siRNA from Dharmacon. In some aspects, any of the p75NTR modulators disclosed herein can be used in any of the disclosed methods.
Any of the disclosed p75NTR modulators can be formulated in a composition, specifically a pharmaceutical composition. Pharmaceutical compositions can comprise a p75NTR modulator and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, from about 0.01 to about 0.1 M and preferably 0.05M phosphate buffer or 0.8% saline. Such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions.
Examples of non-aqueous solvents suitable for use in the presently disclosed subject matter include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
Aqueous carriers suitable for use in the presently disclosed subject matter include, but are not limited to, water, ethanol, alcoholic/aqueous solutions, glycerol, emulsions or suspensions, including saline and buffered media. Oral carriers can be elixirs, syrups, capsules, tablets and the like.
Liquid carriers suitable for use in the presently disclosed subject matter can be used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compounds. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators.
Liquid carriers suitable for use in the presently disclosed subject matter include, but are not limited to, water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also include an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form comprising compounds for parenteral administration. The liquid carrier for pressurized compounds disclosed herein can be halogenated hydrocarbon or other pharmaceutically acceptable propellent.
Solid carriers suitable for use in the presently disclosed subject matter include, but are not limited to, inert substances such as lactose, starch, glucose, methyl-cellulose, magnesium stearate, dicalcium phosphate, mannitol and the like. A solid carrier can further include one or more substances acting as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier can be a finely divided solid which is in admixture with the finely divided active compound. In tablets, the active compound is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active compound. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
Parenteral carriers suitable for use in the presently disclosed subject matter include, but are not limited to, sodium chloride solution. Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous carriers include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose and the like. Preservatives and other additives can also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
Carriers suitable for use in the presently disclosed subject matter can be mixed as needed with disintegrants, diluents, granulating agents, lubricants, binders and the like using conventional techniques known in the art. The carriers can also be sterilized using methods that do not deleteriously react with the compounds, as is generally known in the art.
Disclosed are methods of treating an ocular disease associated with retinal ischemic injury in a subject using a p75NTR modulator. In some aspects, an ocular disease associated with retinal ischemic injury can be, but are not limited to, diabetic retinopathy, retinopathy of prematurity, or traumatic neuropathy.
In some aspects, disclosed are methods of treating a retinal ischemic injury in a subject using a p75NTR modulator.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
Disclosed are methods of treating an ocular disease associated with retinal ischemic injury in a subject comprising administering to the subject in need thereof a p75NTR modulator. Disclosed are methods of treating a retinal ischemic injury in a subject comprising administering to the subject in need thereof a p75NTR modulator.
In some aspects, ischemic areas on retinal angiogram are reduced. In some aspects, acellular capillaries in the subject's retina are reduced.
In some aspects, the disclosed methods of treating an ocular disease associated with retinal ischemic injury in a subject can further comprise administering to the subject a standard treatment for retinal ischemia. Therefore, disclosed are combination therapies comprising a p75NTR modulator and a known treatment for retinal ischemia. In some aspects, the standard treatment for retinal ischemia is anti-vascular endothelial grown factor (anti-VEGF). In some aspects, a standard treatment for retinal ischemia can be steroids. In some aspects, the standard treatment for retinal ischemia administered in addition to the p75NTR modulator can be administered via the same or a different route of administration from the p75NTR modulator. For example, the p75NTR modulator can be administered via oral gavage and the anti-VEGF can be administered via ocular injection.
In some aspects, the p75NTR modulator is administered via oral gavage, intravitreal injection, or subconjunctival injection.
Disclosed are methods of treating an ocular disease associated with retinal ischemic injury in a subject comprising administering to the subject in need thereof a p75NTR modulator, wherein the subject is not diabetic. In other words, in some aspects, the ocular disease associated with retinal ischemic injury is not diabetes.
In some aspects, a p75NTR modulator results in a decrease in p75NTR activity, p75NTR mRNA levels, or p75NTR protein levels. Therefore, in some aspects, p75NTR activity, p75NTR mRNA levels, or p75NTR protein levels can be tested in a subject receiving a p75NTR modulator. Dosing of the p75NTR modulator can be increased or decreased based on the results of the subject's p75NTR activity, p75NTR mRNA levels, and/or p75NTR protein levels.
Not only can a p75NTR modulator be directly administered to a subject in need thereof either alone or formulated as a pharmaceutical composition, but a p75NTR modulator can be delivered to one or more cells, in vitro or ex vivo, and then the one or more cells can be administered to a subject in need thereof. In some aspects, the cell is a mesenchymal stem cell (MSC).
Disclosed are methods of treating an ocular disease associated with retinal ischemic injury in a subject comprising administering to the subject in need thereof one or more mesenchymal stem cells (MSCs) comprising decreased p75NTR expression or activity.
Disclosed are methods of treating retinal ischemic injury in a subject comprising administering to the subject in need thereof one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, the decreased p75NTR expression is a decrease in p75NTR mRNA expression due to the p75NTR modulator. For example, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, the decreased p75NTR expression or activity in the one or more MSCs comprising decreased p75NTR expression or activity is due to a decrease in protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity in the one or more MSCs without affecting p75NTR protein levels in the one or more MSCs.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are treated with a p75NTR modulator. In some aspects, the p75NTR modulator causes a decrease in p75NTR activity or expression in the MSCs. In some aspects, the decrease in p75NTR activity or expression is compared to MSCs not treated with a p75NTR modulator.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are tested for p75NTR activity. In some aspects, only those MSCs with p75NTR activity of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR activity of 25% to 75% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 25% to 75% of original activity are administered to the subject.
In some aspects, neither a pharmacologic therapy or a cell therapy is used, but instead a MSC secretome is used as a therapeutic.
Disclosed are methods of treating an ocular disease associated with retinal ischemic injury in a subject comprising administering to the subject in need thereof a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
Disclosed are methods of treating retinal ischemic injury in a subject comprising administering to the subject in need thereof a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from contacting the MSC with a p75NTR modulator. Thus, in some aspects, the methods further comprise a step of contacting a MSC with a p75NTR modulator prior to collecting the MSC secretome. In some aspects, the MSC secretome used in the disclosed methods is collected from MSCs previously contacted with a p75NTR modulator. In some aspects, the MSC is in culture and the MSC secretome is collected from culture medium. Thus, in some aspects, contacting the MSC with a p75NTR modulator can comprise adding a p75NTR modulator to a cell culture comprising one or more MSCs.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from a genetically modified MSC to decrease expression or activity of p75NTR in the MSC. In some aspects, genetically modified MSCs having decreased expression or activity of p75NTR comprise a reduction or a knock down mutation of p75NTR in the MSCs.
In some aspects, the decreased p75NTR activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity without affecting p75NTR protein levels.
In some aspects, prior to administering the subject the MSC secretome, the MSC secretome is tested for the presence of nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and/or stromal derived factor (SDF-1). In some aspects, only those MSC secretomes with one or more of NGF, VEGF, and SDF-1 are administered to the subject.
Disclosed are methods of improving visual acuity in a subject having a retinal ischemic injury.
In some aspects, the disclosed methods comprise a decrease in p75NTR. In some aspects, a p75NTR modulator can be administered to a subject, one or more MSCs comprising decreased p75NTR expression or activity can be administered to a subject, or a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity can be administered to a subject.
In some aspects, any of the disclosed p75NTR modulators can be used.
Disclosed are methods of improving visual acuity in a subject having a retinal ischemic injury comprising administering to the subject in need thereof a p75NTR modulator.
In some aspects, a subject having a retinal ischemic injury is a subject having an ocular disease associated with retinal ischemic injury. In some aspects, an ocular disease associated with retinal ischemic injury can be, but are not limited to, diabetic retinopathy, retinopathy of prematurity, or traumatic neuropathy.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
In some aspects, improving visual acuity can be due to the p75NTR modulator reducing ischemic areas on retinal angiograms. In some aspects, improving visual acuity can be due to the p75NTR modulator reducing acellular capillaries in the subject's retina.
In some aspects, the p75NTR modulator is administered via oral gavage, intravitreal injection, or subconjunctival injection.
In some aspects, the disclosed methods of improving visual acuity in a subject having a retinal ischemic injury comprising administering to the subject in need thereof a p75NTR modulator can further comprise administering to the subject a standard treatment for retinal ischemia. Therefore, disclosed are combination therapies comprising a p75NTR modulator and a known treatment for retinal ischemia. In some aspects, the standard treatment for retinal ischemia is anti-vascular endothelial grown factor (anti-VEGF). In some aspects, a standard treatment for retinal ischemia can be steroids. In some aspects, the standard treatment for retinal ischemia administered in addition to the p75NTR modulator can be administered via the same or a different route of administration from the p75NTR modulator. For example, the p75NTR modulator can be administered via oral gavage and the anti-VEGF can be administered via ocular injection.
Disclosed are methods of improving visual acuity in a subject having a retinal ischemic injury comprising administering to the subject in need thereof a p75NTR modulator, wherein the subject is not diabetic. In other words, in some aspects, the ocular disease associated with retinal ischemic injury is not diabetes.
Not only can a p75NTR modulator be directly administered to a subject in need thereof, either alone or formulated as a pharmaceutical composition, but a p75NTR modulator can be delivered to one or more cells, in vitro, and then the one or more cells can be administered to a subject in need thereof. In some aspects, the cell is a mesenchymal stem cell (MSC).
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
Disclosed are methods of improving visual acuity in a subject having a retinal ischemic injury comprising administering to the subject in need thereof one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, the decreased p75NTR expression is a decrease in p75NTR mRNA expression due to the p75NTR modulator. For example, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, the decreased p75NTR expression or activity in the one or more MSCs comprising decreased p75NTR expression or activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity in the one or more MSCs without affecting p75NTR protein levels in the one or more MSCs.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are treated with a p75NTR modulator. In some aspects, the p75NTR modulator causes a decrease in p75NTR activity or expression in the MSCs. In some aspects, the decrease in p75NTR activity or expression is compared to MSCs not treated with a p75NTR modulator.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are tested for p75NTR activity. In some aspects, only those MSCs with p75NTR activity of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR activity of 25% to 75% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 25% to 75% of original activity are administered to the subject.
In some aspects, neither a pharmacologic therapy or a cell therapy is used, but instead a MSC secretome is used as a therapeutic.
Disclosed are methods of improving visual acuity in a subject having a retinal ischemic injury comprising administering to the subject a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from contacting the MSC with a p75NTR modulator. Thus, in some aspects, the methods further comprise a step of contacting a MSC with a p75NTR modulator prior to collecting the MSC secretome. In some aspects, the MSC secretome used in the disclosed methods is collected from MSCs previously contacted with a p75NTR modulator. In some aspects, the MSC is in culture and the MSC secretome is collected from culture medium. Thus, in some aspects, contacting the MSC with a p75NTR modulator can comprise adding a p75NTR modulator to a cell culture comprising one or more MSCs.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from a genetically modified MSC to decrease expression or activity of p75NTR in the MSC. In some aspects, genetically modified MSCs having decreased expression or activity of p75NTR comprise a reduction or a knock down mutation of p75NTR in the MSCs.
In some aspects, the decreased p75NTR activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity without affecting p75NTR protein levels.
In some aspects, prior to administering the subject the MSC secretome, the MSC secretome is tested for the presence of NGF, VEGF, and/or SDF-1. In some aspects, only those MSC secretomes with one or more of NGF, VEGF, and/or SDF-1 are administered to the subject.
Disclosed are methods of preventing ischemia-induced retinal capillary degeneration in a subject.
In some aspects, the disclosed methods comprise a decrease or inhibition of p75NTR. In some aspects, a p75NTR modulator can be administered to a subject, one or more MSCs comprising decreased p75NTR expression or activity can be administered to a subject, or a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity can be administered to a subject.
In some aspects, any of the disclosed p75NTR modulators can be used.
Disclosed are methods of preventing ischemia-induced retinal capillary degeneration in a subject comprising administering to the subject in need thereof a p75NTR modulator, wherein the p75NTR modulator inhibits the expression or activity of p75NTR in mesenchymal stem cells (MSCs) in the retina of the subject.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
In some aspects, the p75NTR modulator can reduce ischemic areas on retinal angiograms. In some aspects, the p75NTR modulator can reduce acellular capillaries in the subject's retina.
In some aspects, the p75NTR modulator is administered via oral gavage, intravitreal injection, or subconjunctival injection.
Not only can a p75NTR modulator be directly administered to a subject in need thereof, either alone or formulated as a pharmaceutical composition, but a p75NTR modulator can be delivered to to one or more cells, in vitro, and then the one or more cells can be administered to a subject in need thereof. In some aspects, the cell is a mesenchymal stem cell (MSC).
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
Disclosed are methods of preventing ischemia-induced retinal capillary degeneration in a subject comprising administering to the subject one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, the decreased p75NTR expression is a decrease in p75NTR mRNA expression due to the p75NTR modulator. For example, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, the decreased p75NTR expression or activity in the one or more MSCs comprising decreased p75NTR expression or activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity in the one or more MSCs without affecting p75NTR protein levels in the one or more MSCs.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are treated with a p75NTR modulator. In some aspects, the p75NTR modulator causes a decrease in p75NTR activity or expression in the MSCs. In some aspects, the decrease in p75NTR activity or expression is compared to MSCs not treated with a p75NTR modulator.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are tested for p75NTR activity. In some aspects, only those MSCs with p75NTR activity of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR activity of 25% to 75% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 25% to 75% of original activity are administered to the subject.
In some aspects, neither a pharmacologic therapy or a cell therapy is used, but instead a MSC secretome is used as a therapeutic.
Disclosed are methods preventing ischemia-induced retinal capillary degeneration in a subject comprising administering to the subject a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from contacting the MSC with a p75NTR modulator. Thus, in some aspects, the methods further comprise a step of contacting a MSC with a p75NTR modulator prior to collecting the MSC secretome. In some aspects, the MSC secretome used in the disclosed methods is collected from MSCs previously contacted with a p75NTR modulator. In some aspects, the MSC is in culture and the MSC secretome is collected from culture medium. Thus, in some aspects, contacting the MSC with a p75NTR modulator can comprise adding a p75NTR modulator to a cell culture comprising one or more MSCs.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from a genetically modified MSC to decrease expression or activity of p75NTR in the MSC. In some aspects, genetically modified MSCs having decreased expression or activity of p75NTR comprise a reduction or a knock down mutation of p75NTR in the MSCs.
In some aspects, the decreased p75NTR activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity without affecting p75NTR protein levels.
In some aspects, prior to administering the subject the MSC secretome, the MSC secretome is tested for the presence of NGF, VEGF and/or SDF-1. In some aspects, only those MSC secretomes with one or more of NGF, VEGF, and SDF-1 are administered to the subject.
Disclosed are methods of increasing vascular protection in an ischemic retina of a subject.
In some aspects, the disclosed methods comprise a decrease in p75NTR. In some aspects, a p75NTR modulator can be administered to a subject, a MSC comprising decreased p75NTR expression or activity can be administered to a subject, or a MSC secretome collected from a MSC comprising decreased p75NTR expression or activity can be administered to a subject.
In some aspects, any of the disclosed p75NTR modulators can be used.
Disclosed are methods of increasing vascular protection in an ischemic retina of a subject comprising administering to the retina of the subject in need thereof a p75NTR modulator, wherein the p75NTR modulator inhibits expression or activity of p75NTR in MSCs in the retina of the subject.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
In some aspects, the p75NTR modulator can reduce ischemic areas on retinal angiograms. In some aspects, the p75NTR modulator can reduce acellular capillaries in the subject's retina.
In some aspects, the p75NTR modulator reduces p75NTR expression. In some aspects, the reduced or decreased p75NTR expression is a decrease in p75NTR mRNA expression due to the p75NTR modulator. For example, a p75NTR-specific siRNA can reduce or decrease p75NTR mRNA expression.
In some aspects, the p75NTR modulator is administered via oral gavage, intravitreal injection, or subconjunctival injection.
Not only can a p75NTR modulator be directly administered to a subject in need thereof, either alone or formulated as a pharmaceutical composition, but a p75NTR modulator can be delivered to one or more cells, in vitro, and then the one or more cells can be administered to a subject in need thereof. In some aspects, the cell is a mesenchymal stem cell (MSC).
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
Disclosed are methods of increasing vascular protection in an ischemic retina of a subject comprising administering to the retina of the subject one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, the decreased p75NTR expression is a decrease in p75NTR mRNA expression due to the p75NTR modulator. For example, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, the decreased p75NTR activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity without affecting p75NTR protein levels.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are treated with a p75NTR modulator. In some aspects, the p75NTR modulator causes a decrease in p75NTR activity or expression in the MSCs. In some aspects, the decrease in p75NTR activity or expression is compared to MSCs not treated with a p75NTR modulator.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are tested for p75NTR activity. In some aspects, only those MSCs with p75NTR activity of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR activity of 25% to 75% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 25% to 75% of original activity are administered to the subject.
In some aspects, neither a pharmacologic therapy or a cell therapy is used, but instead a MSC secretome is used as a therapeutic.
Disclosed are methods of increasing vascular protection in an ischemic retina of a subject comprising administering to the retina of the subject a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from contacting the MSC with a p75NTR modulator. Thus, in some aspects, the methods further comprise a step of contacting a MSC with a p75NTR modulator prior to collecting the MSC secretome. In some aspects, the MSC secretome used in the disclosed methods is collected from MSCs previously contacted with a p75NTR modulator. In some aspects, the MSC is in culture and the MSC secretome is collected from culture medium. Thus, in some aspects, contacting the MSC with a p75NTR modulator can comprise adding a p75NTR modulator to a cell culture comprising one or more MSCs.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from a genetically modified MSC to decrease expression or activity of p75NTR in the MSC. In some aspects, genetically modified MSCs having decreased expression or activity of p75NTR comprise a reduction or a knock down mutation of p75NTR in the MSCs.
In some aspects, the decreased p75NTR activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity without affecting p75NTR protein levels.
In some aspects, prior to administering the subject the MSC secretome, the MSC secretome is tested for the presence of NGF, VEGF, and/or SDF-1. In some aspects, only those MSC secretomes with one or more of NGF, VEGF, and SDF-1 are administered to the subject.
Disclosed are methods of increasing vascular homing of MSCs at a site of ischemic injury in a subject.
In some aspects, the disclosed methods comprise a decrease in p75NTR. In some aspects, a p75NTR modulator can be administered to a subject, one or more MSCs comprising decreased p75NTR expression or activity can be administered to a subject, or a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity can be administered to a subject.
In some aspects, any of the disclosed p75NTR modulators can be used.
Disclosed are methods of increasing vascular homing of MSCs at a site of ischemic injury in a subject comprising administering to the subject in need thereof a p75NTR modulator, wherein the p75NTR modulator inhibits expression or activity of p75NTR in the MSCs. In some aspects, the site of ischemic injury is in the retina of the subject.
In some aspects, the concentration of MSCs are increased in the retinal capillaries at the site of the ischemic injury in the subject's retina. In other words, the number of MSCs increase in the retinal capillaries at the site of the ischemic injury in the subject's retina in response to administering a p75NTR modulator to the subject.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
In some aspects, the p75NTR modulator reduces p75NTR expression. In some aspects, the reduced or decreased p75NTR expression is a decrease in p75NTR mRNA expression due to the p75NTR modulator. For example, a p75NTR-specific siRNA can reduce or decrease p75NTR mRNA expression.
In some aspects, the p75NTR modulator is administered via oral gavage, intravitreal injection, or subconjunctival injection.
Not only can a p75NTR modulator be directly administered to a subject in need thereof, either alone or
Not only can a p75NTR modulator be directly administered to a subject in need thereof, either alone or formulated as a pharmaceutical composition, but a p75NTR modulator can be delivered to to one or more cells, in vitro, and then the one or more cells can be administered to a subject in need thereof. In some aspects, the cell is a mesenchymal stem cell (MSC).as a pharmaceutical composition, but a p75NTR modulator can be delivered to a cell, in vitro, and then the cell can be administered to a subject in need thereof.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
Disclosed are methods of increasing vascular homing of MSCs at a site of ischemic injury in a subject comprising administering to the subject one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, the decreased p75NTR expression is a decrease in p75NTR mRNA expression due to the p75NTR modulator. For example, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, the decreased p75NTR activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity without affecting p75NTR protein levels.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are treated with a p75NTR modulator. In some aspects, the p75NTR modulator causes a decrease in p75NTR activity or expression in the MSCs. In some aspects, the decrease in p75NTR activity or expression is compared to MSCs not treated with a p75NTR modulator.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are tested for p75NTR activity. In some aspects, only those MSCs with p75NTR activity of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR activity of 25% to 75% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 25% to 75% of original activity are administered to the subject.
In some aspects, neither a pharmacologic therapy or a cell therapy is used, but instead a MSC secretome is used as a therapeutic.
Disclosed are methods of increasing vascular homing of MSCs at a site of ischemic injury in a subject comprising administering to the subject a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from contacting the MSC with a p75NTR modulator. Thus, in some aspects, the methods further comprise a step of contacting a MSC with a p75NTR modulator prior to collecting the MSC secretome. In some aspects, the MSC secretome used in the disclosed methods is collected from MSCs previously contacted with a p75NTR modulator. In some aspects, the MSC is in culture and the MSC secretome is collected from culture medium. Thus, in some aspects, contacting the MSC with a p75NTR modulator can comprise adding a p75NTR modulator to a cell culture comprising one or more MSCs.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from a genetically modified MSC to decrease expression or activity of p75NTR in the MSC. In some aspects, genetically modified MSCs having decreased expression or activity of p75NTR comprise a reduction or a knock down mutation of p75NTR in the MSCs.
In some aspects, the decreased p75NTR activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity without affecting p75NTR protein levels.
In some aspects, prior to administering the subject the MSC secretome, the MSC secretome is tested for the presence of NGF, VEGF, and/or SDF-1. In some aspects, only those MSC secretomes with one or more of NGF, VEGF, and SDF-1 are administered to the subject.
Disclosed are methods of altering a MSC's secretome comprising inhibiting the expression or activity of p75NTR in the MSC. In some aspects, altering a MSC's secretome can be improving a MSC's secretome. In some aspects, an improved secretome can be a secretome comprising increased levels of NGF, VEGF, and/or SDF-1 compared to a secretome from non-treated MSC cells. In some aspects, an increase of at least 1.5 to 2-fold can be an improved secretome.
In some aspects, inhibiting the expression or activity of p75NTR in the MSC comprises contacting the MSC with a p75NTR modulator. In some aspects, the MSC is in culture. In some aspects, the MSC is in a subject. Thus, in some aspects, contacting the MSC with a p75NTR modulator can comprise adding a p75NTR modulator to a cell culture comprising one or more MSCs. In some aspects, contacting the MSC with a p75NTR modulator can comprise administering a p75NTR modulator to a subject, wherein the subject comprises MSCs that can come in contact with the p75NTR modulator. In some aspects, the MSCs are in culture. In some aspects, the MSCs are in a retina of a subject. Therefore, the disclosed methods can be performed in vitro or in vivo.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
In some aspects, inhibiting the expression or activity of p75NTR in MSCs comprises genetically modifying the MSCs to decrease expression or activity of p75NTR in the MSCs. In some aspects, genetically modifying the MSCs to have decreased expression or activity of p75NTR comprises a reduction or a knock down mutation of p75NTR in the MSCs.
In some aspects, expression of one or more of VEGF, SDF-1α, and NGF in the MSC is increased. In some aspects, one or more of VEGF, SDF-1α, and NGF is increased in the MSC secretome. For example, if the disclosed method is performed in vitro, one or more of VEGF, SDF-1α, and NGF is increased in the cell culture medium.
Disclosed are methods of increasing survival factors in a HRE comprising contacting the HRE with a MSC having decreased p75NTR expression or activity. In some aspects, levels of VEGF-A, Akt, and/or Bcl-2 in the HRE are increased.
In some aspects, contacting the HRE with the MSC having decreased p75NTR expression or activity comprises first contacting the MSC with a p75NTR modulator. In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
Disclosed are methods of increasing survival factors in a HRE comprising contacting the HRE with a MSC secretome, wherein the MSC has decreased p75NTR expression or activity. In some aspects, levels of VEGF-A, Akt, and/or Bcl-2 in the HRE are increased in response to the MSC secretome. In some aspects, the MSC has decreased p75NTR expression or activity due to contacting the MSC with a p75NTR modulator. In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
In some aspects, contacting the HRE with a MSC secretome, wherein the MSC has decreased p75NTR expression or activity, can comprise adding the MSC secretome to a cell culture comprising one or more HREs. In some aspects, contacting the HRE with a MSC secretome, wherein the MSC has decreased p75NTR expression or activity, can comprise administering a MSC secretome, from a MSC having decreased p75NTR expression or activity, to a subject, wherein the subject comprises HREs that will come in contact with the MSC secretome. In some aspects, the HREs are in culture. In some aspects, the HREs are in a retina of a subject. Therefore, the disclosed methods can be performed in vitro or in vivo.
Disclosed are methods of increasing angiogenic effects of a HRE comprising contacting the HRE with a MSC having decreased p75NTR expression or activity, wherein HRE migration is increased.
Disclosed are methods of increasing angiogenic effects of a HRE comprising contacting the HRE with a MSC secretome, wherein the MSC has decreased p75NTR expression or activity, wherein HRE migration is increased.
In some aspects, the HRE is in culture. In some aspects, the HRE is in a subject. In some aspects, contacting the HRE with one or more MSCs having decreased p75NTR expression or activity comprises first contacting the MSC with a p75NTR modulator. In some aspects, the decreased expression or activity of p75NTR in MSCs comprises genetically modifying the MSCs to decrease expression or activity of p75NTR in the MSCs. In some aspects, genetically modifying the MSCs to have decreased expression or activity of p75NTR comprises a reduction or a knock down mutation of p75NTR in the MSCs.
In some aspects, contacting the HRE with a MSC secretome, wherein the MSC has decreased p75NTR expression or activity, can comprise administering a MSC having decreased p75NTR expression or activity to a subject, wherein the subject comprises HREs that can come in contact with the MSC. In some aspects, the HREs are in culture. In some aspects, the MSCs are in a retina of a subject. Therefore, the disclosed methods can be performed in vitro or in vivo.
In some aspects of the disclosed methods, the ability of the HRE to form tubes is increased. In other words, capillaries can be formed which increases the angiogenic effect. In some aspects, one or more HREs comprise a tube, and wherein the tube length is increased in the HRE. The increase in tube length can be compared to an HRE not contacted with a MSC having decreased p75NTR expression or activity or with a MSC secretome from a MSC having decreased p75NTR expression or activity.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
Disclosed are methods of administering a p75NTR modulator to a subject having diabetes. In some aspects, the subject having diabetes has advanced stage diabetes. In some aspects, advanced stage diabetes can also be known as advanced stage of diabetic complications. Advanced stage of diabetic complications can be identified by an altered (increased) ratio of albumin excretion to creatinine and low glomerular filtration rate (GFR). Histopathological changes (e.g. glomerular size and PASH stain) can also be identified. For example, late stage, or advanced stage, diabetes can be identified by diabetic kidney disease progression to renal failure. In some aspects, diabetes can cause microvascular complications such as diabetic retinopathy (eye), nephropathy (kidney) that can progress to end stage renal disease (ESRD) and renal failure.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
Disclosed are methods of decreasing urinary albumin excretion rate in a subject having diabetes comprising administering to the subject a p75NTR modulator.
Disclosed are methods of decreasing inflammation in the kidney of a subject having diabetes comprising administering to the subject a p75NTR modulator. In some aspects, the presence of IL-1β is decreased in the kidney or urine of the subject and/or the presence of IL-6 is decreased in the kidney of the subject and/or the presence of TNF-α is decreased in the kidney of the subject and/or the presence of NGF is increased in the kidney of the subject. The presence or absence of one or more of these, as described herein, is indicative of a decrease in inflammation in the kidney. In some aspects, IL-1β, TNF-α, and/or IL-6 are detected in the urine or blood of a subject. Because the kidney cannot necessarily be accessed in a live subject, the presence of IL-1β, TNF-α, and/or IL-6 in the urine or blood of the subject can be indicative of increased levels in the kidney.
Disclosed are methods of decreasing IL-1β in a subject having diabetes comprising administering to the subject a p75NTR modulator, wherein IL-1β is decreased in one or both kidneys after administration of the p75NTR modulator. In some aspects, a decrease in IL-1β is indicative of a decrease in inflammation in the kidney of the subject. In some aspects, the decrease in IL-10 is a decrease in mRNA levels, protein levels, or protein activity. In some aspects, the levels or activity of IL-1β is detected in the urine of a subject and correlates to kidney levels or activity.
Disclosed are methods of decreasing IL-6 in a subject having diabetes comprising administering to the subject in need thereof a p75NTR modulator, wherein IL-6 is decreased in one or both kidneys after administration of the p75NTR modulator. In some aspects, a decrease in IL-6 is indicative of a decrease in inflammation in the kidney of the subject. In some aspects, the decrease in IL-6 is a decrease in mRNA levels, protein levels, or protein activity. In some aspects, the levels or activity of IL-6 is detected in the urine of a subject and correlates to kidney levels or activity.
Disclosed are methods of decreasing TGF-β in a subject having diabetes comprising administering to the subject in need thereof a p75NTR modulator, wherein TGF-β is decreased in one or both kidneys after administration of the p75NTR modulator. In some aspects, a decrease in TGF-β is indicative of a decrease in inflammation in the kidney of the subject. In some aspects, the decrease in TGF-β is a decrease in mRNA levels, protein levels, or protein activity. In some aspects, the levels or activity of TGF-β is detected in the urine of a subject and correlates to kidney levels or activity.
Disclosed are methods of decreasing one or more markers of fibrosis in a subject having diabetes comprising administering to the subject in need thereof a p75NTR modulator, wherein one or more markers of fibrosis is decreased in one or both kidneys after administration of the p75NTR modulator. In some aspects, the disclosed methods of decreasing one or more markers of fibrosis can be performed in non-diabetic subjects. Thus, also disclosed are methods of decreasing one or more markers of fibrosis in a non-diabetic subject comprising administering to the subject in need thereof a p75NTR modulator, wherein one or more markers of fibrosis is decreased in one or both kidneys after administration of the p75NTR modulator. In some aspects, the markers of fibrosis are one or more of fibronectin or α-SMA. In some aspects, the markers of fibrosis are detected in the urine of a subject and correlates to kidney levels or activity.
Disclosed are methods of decreasing fibrotic tissue in a kidney of a subject having diabetes comprising administering to the subject in need thereof a p75NTR modulator.
Disclosed are methods of treating late stage diabetes in a subject comprising administering to the subject in need thereof a p75NTR modulator.
Not only can a p75NTR modulator be directly administered to a subject in need thereof, either alone or formulated as a pharmaceutical composition, but a p75NTR modulator can be delivered to one or more cells, in vitro, and then the one or more cells can be administered to a subject in need thereof. In some aspects, the cell is a mesenchymal stem cell (MSC).
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, the p75NTR modulator can be a p75NTR-specific siRNA.
Instead of administering a p75NTR modulator to a subject having diabetes in any of the disclosed methods, the methods can comprise administering to the subject having diabetes one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, the decreased p75NTR expression is a decrease in p75NTR mRNA expression due to the p75NTR modulator. For example, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, the decreased p75NTR activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity without affecting p75NTR protein levels.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are treated with a p75NTR modulator. In some aspects, the p75NTR modulator causes a decrease in p75NTR activity or expression in the MSCs. In some aspects, the decrease in p75NTR activity or expression is compared to MSCs not treated with a p75NTR modulator.
In some aspects, prior to administering the subject the one or more MSCs comprising decreased p75NTR expression or activity, the MSCs are tested for p75NTR activity. In some aspects, only those MSCs with p75NTR activity of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR activity of 25% to 75% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 50% of original activity are administered to the subject. In some aspects, only those MSCs with p75NTR expression levels of 25% to 75% of original activity are administered to the subject.
In some aspects, neither a pharmacologic therapy or a cell therapy is used, but instead a MSC secretome is used as a therapeutic.
Instead of administering a p75NTR modulator or a cell therapy to a subject having diabetes in any of the disclosed methods, the methods can comprise administering to the subject a MSC secretome collected from one or more MSCs comprising decreased p75NTR expression or activity.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from contacting the MSC with a p75NTR modulator. Thus, in some aspects, the methods further comprise a step of contacting a MSC with a p75NTR modulator prior to collecting the MSC secretome. In some aspects, the MSC secretome used in the disclosed methods is collected from MSCs previously contacted with a p75NTR modulator. In some aspects, the MSC is in culture and the MSC secretome is collected from culture medium. Thus, in some aspects, contacting the MSC with a p75NTR modulator can comprise adding a p75NTR modulator to a cell culture comprising one or more MSCs.
In some aspects, any of the p75NTR modulators disclosed herein can be used in the disclosed methods. For example, the p75NTR modulator can be LM11A-31 or a derivative thereof. In some aspects, a p75NTR modulator can be a p75NTR-specific siRNA which can reduce or decrease p75NTR mRNA expression.
In some aspects, a MSC comprising decreased p75NTR expression or activity results from a genetically modified MSC to decrease expression or activity of p75NTR in the MSC. In some aspects, genetically modified MSCs having decreased expression or activity of p75NTR comprise a reduction or a knock down mutation of p75NTR in the MSCs.
In some aspects, the decreased p75NTR activity is due to a decrease in p75NTR protein levels or amounts. In some aspects, the decreased p75NTR activity is due to a blocking of p75NTR activity without affecting p75NTR protein levels.
In some aspects, prior to administering the subject the MSC secretome, the MSC secretome is tested for the presence of NGF, VEGF, and/or SDF-1. In some aspects, only those MSC secretomes with one or more of NGF, VEGF, and SDF-1 are administered to the subject.
The presently disclosed subject matter discloses methods of administering p75NTR modulators to a subject or administering p75NTR modulators to a cell, wherein the cell is administered to a subject. The method can comprise the step of administering to a subject an effective amount of a composition comprising a p75NTR modulator, such as any of the compositions disclosed herein.
As used herein, administering can be effected or performed using any of the various methods known to those skilled in the art. The composition can be administered, for example, subcutaneously, intravenously, parenterally, intraperitoneally, intradermally, intramuscularly, topically, enteral (e.g., orally), rectally, nasally, buccally, sublingually, vaginally, by inhalation spray, by drug pump or via an implanted reservoir in dosage formulations containing conventional non-toxic, physiologically acceptable carriers or vehicles. In some aspects, the composition can be administered via oral gavage.
Further, the presently disclosed compositions can be administered to a localized area in need of treatment. This can be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, transdermal patches, by injection, by catheter, by suppository, or by implant (the implant can optionally be of a porous, non-porous, or gelatinous material), including membranes, such as sialastic membranes or fibers.
The form in which the composition is administered (e.g., syrup, elixir, capsule, tablet, solution, foams, emulsion, gel, sol) will depend in part on the route by which it is administered. For example, for mucosal (e.g., oral mucosa, rectal, intestinal mucosa, bronchial mucosa) administration, nose drops, aerosols, inhalants, nebulizers, eye drops or suppositories can be used. The composition can also be used to coat bioimplantable materials to enhance neurite outgrowth, neural survival, or cellular interaction with the implant surface. The compositions and agents disclosed herein can be administered together with other biologically active agents, such as analgesics, anti-inflammatory agents, anesthetics and other agents which can control one or more symptoms or causes of a p75NTR-mediated condition.
Additionally, administration can comprise administering to the subject a plurality of dosages over a suitable period of time. Such administration regimens can be determined according to routine methods, upon a review of the instant disclosure.
Cell therapy in general and mesenchymal stem cells (MSCs) can be an attractive candidate for retinal regeneration. MSCs are multipotent stem cells present in adult marrow and have the potential to differentiate into lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle and marrow stroma. One advantage of adult MSCs is that they can be isolated from the bone marrow, peripheral blood, or adipose tissue of a subject in reasonable quantity; however, the in vitro expansion of MSCs, a lengthy process, prior to administration, can result in a loss of stemness and can be hampered by the age and the disease state of the patient. MSCs are perceived as “immune-privileged” cells and as such are widely used clinically in allogeneic settings. MSCs are home to sites of inflammation where they secrete a variety of soluble factors, including growth factors, cytokines, and chemokines. While the secretion of paracrine factors by MSCs is well-established as a reparative mechanism, the engrafting of MSCs to injured tissues has not been proven to be a perquisite for the reparative action. Animal studies demonstrated that the subretinal transplantation of MSCs delays retinal neurodegeneration and preserves neural retinal function; however, the vascular protection of MSCs transplanted in the ischemic retina has not been fully elucidated.
Genetic deletion of p75NTR prevents diabetes-induced degeneration of retinal capillaries identified by the presence of acellular capillaries. Based on this similarity between ischemia/reperfusion (I/R) and diabetic retinopathy, the vascular protective effects of p75NTR genetic deletion was examined in acute retinal ischemic/reperfusion (I/R) injury. Trypsin-digested retinas showed that I/R resulted in a significant increase in the number of acellular capillaries (3-Fold) in WT mice, compared to their sham-operated controls, whereas the sham group showed a mean of 2.51±0.5 acellular capillaries, as compared to the I/R group, showing a mean of 7.06±0.83 acellular capillaries/field (
MSCs are widely used; however, whether the incorporation and engrafting of MSCs to injured tissues is required to exert their vascular protection remains to be elucidated. GFP-labelled MSCs that could be traced post intravitreal injection were used. I/R was performed and, two-days later, GFP-MSCs were intravitreally injected into WT ischemic retinas. The homing and integration of MSCs into ischemic vasculature were followed after an additional 3 days (
Since the p75NTR has been shown to possibly inhibit differentiation of MSCS into multiple cell types, the impact of silencing p75NTR on MSCs homing and engrafting into ischemic retinas was examined. Optimization experiments showed that 100 nM of siRNA against p75NTR was as effective as 300 nM of siRNA to reduce p75NTR mRNA expression. To assess the change in vascular homing upon knocking-down p75NTR expression, the Scr- or Si-100-treated GFP-labeled MSCs were injected intravitreally, 2 days post I/R injury in WT mice (
Secretion of paracrine factors by MSCs is well-established as their primary reparative mechanism. Thus, the impact of modulating p75NTR on the MSCs secretome was characterized. As shown in
Since an improved MSC secretome was observed by silencing p75NTR expression on MSCs (
The angiogenic behavior of HREs showed significant improvement upon treatment with CM of p75NTR-silenced MSCs, where HREs migration significantly increased by 1.4 Fold after 12 h of treatment (
LM11A-31, the pharmacologic modulator of p75NTR receptor, showed similar results to p75NTR genetic silencing in improving the secretome of MSCs. As shown in
CM of p75NTR-modulated MSCs using LM11A-31 showed similar enhanced angiogenic response in HREs (
As shown in
Retinal ischemia is a common underlying pathology for multiple retinal diseases, including diabetic retinopathy, retinopathy of prematurity, traumatic optic neuropathy and acute closed angle glaucoma. Vascular cell death and development of acellular capillaries are well-accepted surrogate markers for retinal ischemia. Stem cell therapy, in general, and mesenchymal stem cells (MSCs), in particular, have demonstrated hopeful results to improve ischemic and neuronal disorders. It has been demonstrated that increases in the neurotrophin receptor p75NTR are closely linked to retinal inflammation, barrier dysfunction and the development of acellular capillaries. Here, it is demonstrated that the vascular protective effects of modulating p75NTR and MSCs injection using an acute model of retinal ischemia/reperfusion in mice. The main findings of the current study are: (1) the vascular protective effects of p75NTR deletion against retinal ischemia is enhanced by MSCs injection; (2) silencing p75NTR expression on MSCs enhanced their vascular homing and potentiated the vascular protection against I/R-injury; (3) modulating p75NTR on MSCs using siRNA or pharmacologically using LM11A-31 enriched their secretome with trophic and angiogenic factors, such as NGF, SDF-1, and VEGF; (4) conditioned medium of p75NTR modulated MSCs stimulated the survival and angiogenic behavior of retinal endothelial cells; (5) further intervention with LM11A-31 significantly improved decline in visual acuity post retinal ischemic injury.
P75NTR signal transduction pathways are extremely variable because they are dependent on cell type, cell differentiation status, neurotrophin binding, interacting transmembrane co-receptor as well as the availability of intracellular adaptor molecule. This leads to divergent cellular responses, including progenitor differentiation, cell survival, apoptosis, and cell migration and invasion. These findings showed that transient exposure to I/R resulted in a significant increase in acellular capillaries formation in WT mice that was ameliorated in p75NTR−/− mice (
Capillary degeneration associated with p75NTR signaling may be due to failure of endothelial hematopoietic stem cells to maintain normal retinal vasculature or to repair damaged vasculature. The P75NTR receptor, also known as CD271, enriches several progenitor/stem cells subtypes, including MSCs. P75NTR is involved in MSCs differentiation where expression of p75NTR was shown to rapidly downregulate upon differentiation of MSCs in vitro. Moreover, p75NTR could directly hinder the differentiation of MSCs through the inhibition of transcription factors, including Runx2 and OSX, which are essential for osteoblast differentiation and for the expression of chondrogenesis marker; Sox9 and the myogenic marker, Myf5. The transplantation of bone marrow-derived MSCs was shown to rescue photoreceptor cells in dystrophic retina of rhodopsin knockout mice, indicating a therapeutic benefit in retinitis pigmentosa. The neuroprotection of MSCs has been reported in rat retina subjected to I/R, where injection of MSCs preserved number of RGCs compared to controls. While MSCs-associated neuroprotection has been demonstrated, this study explored the MSCs-mediated vascular protection in ischemic retina. The intravitreal injection of MSCs 2 days post I/R injury exerted vascular protection in both WT and p75NTR−/− mice with enhanced homing and integration into retinal capillaries (
To explore the possible behavior of MSCs post injection and its interaction with retinal microvasculature, a large number of MSCs (100,000 cells/eye) were injected. It was interesting to observe different behavior at different time points (
To investigate the underlying mechanism behind the augmented vascular protection observed after silencing p75NTR expression on MSCs, their CM were examined for secreted trophic and growth factors. The silencing of MSCs-p75NTR enriched their CM with VEGF, NGF and SDF-1α (
Of note, changes in the gene expression of both survival and apoptotic markers were explored in HREs upon treatment with the CM of p75NTR-silenced MSCs. Conventionally, the Bcl-2/Bax axis regulates mitochondrial-induced apoptosis in endothelial cells through the reciprocation of the anti-apoptotic proteins; Bcl-2 and the pro-apoptotic protein; Bax, which might result in a neutral net effect. Remarkably, marked increases in the expression of both Bcl-2 and Bax in HREs treated with CM of p75NTR-silenced MSCs was observed. Although the pro-apoptotic marker BAX was increased, other pro-apoptotic markers, such as p53 and caspase-3 transcripts, did not show significant difference from the controls (
In parallel to genetic silencing of MSCs-p75NTR data, pharmacological inhibition of MSCs-p75NTR using LM11A-31 enriched MSCs secretome with SDF-1α, NGF and VEGFA and augmented the paracrine angiogenic behavior on endothelial cells in vitro (
Among stem cells including embryonic, neural and adult stem cells, MSCs gained interest because they can be obtained from the patients' bone marrow in quantities appropriate for clinical application. Since the expansion of MSCs from each patient can be possibly hampered by the disease state, allogenic cell therapy will be the way to go. Here, evidence is provided that combination of p75NTR modulation strategy and MSCs injection can serve as novel therapeutic approach to rescue visual function in ischemic retinal diseases with superior therapeutic potential than solely using MSCs. The underlying mechanism can involve augmented SDF-1, VEGF and NGF signaling pathways. The current findings support the therapeutic benefit of the orally bioavailable compound, LM11A-31 for ischemic retinal diseases. While homing and integration of MSCs into retina vasculature were observed, that process was correlated with vascular protection post-ischemic injury.
All animal experiments were conducted in agreement with Association for Research in Vision and Ophthalmology statement for use of animals in ophthalmic and vision research, and Charlie Norwood VA Medical Center Animal Care and Use Committee (ACORP #16-01-088). The p75NTR, B6.129S4Ngfrtm1Jae (p75NTR−/−, exon III knockout mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed with C57BL6-J mice (Jackson Laboratories, Bar Harbor, Maine, USA). These mice were crossed and back-crossed to establish a colony of homozygous p75NTR−/− and WT breeders that produced the mice used in the current study.
For surgeries, mice were anesthetized with intraperitoneal ketamine (50 mg/kg; Hospira, Inc., Lake Forest, IL, USA) and xylazine (10 mg/kg; Akorn, Decatur, IL, USA). Retinal ischemia/reperfusion (I/R) was performed as described previously in Coucha, M. et al. Modulating Expression of Thioredoxin Interacting Protein (TXNIP) Prevents Secondary Damage and Preserves Visual Function in a Mouse Model of Ischemia/Reperfusion. Int. J. Mol. Sci. 2019, 20. Briefly, pupils were dilated with 1% Atropine Sulfate (Akorn, Inc., Lake Forest, IL, USA). The anterior chamber was cannulated with a 32 gauge needle attached to a line from a saline reservoir at a height calibrated to yield 120 mmHg. The intraocular pressure (IOP) was elevated to 120 mmHg for 45-60 min. I/R injury and choroidal nonperfusion was evident by whitening of the anterior segment of the globe and blanching of the episcleral veins. During infusion, topical anesthesia (0.5% tetracaine HCL, Bausch & Lomb, NJ, USA) was applied to the cornea. After ischemia, the needle was immediately withdrawn, allowing for rapid reperfusion, IOP was normalized, and reflow of the retinal vasculature was confirmed by observation of the episcleral veins. Topical antibiotic (NeoPolycin, Perrigo, Allegan, MI, USA) was applied to the cornea to minimize infection. I/R injury was performed in one eye with the other undergoing sham surgery, in which the needle was inserted into the anterior chamber without elevating the IOP. Mice were sacrificed 10 days post I/R and eyes were processed.
GFP-labeled mouse MSCs were obtained as a kind gift from Dr. William D. Hill (MUSC, Charleston, SC) [57] and used between passages 5-9. MSCs cells were cultured in high glucose DMEM (4.5 g/L D-glucose, Thermo-Fisher Grand Island, NY, USA) supplemented with 10% FBS and 1% penicillin/streptomycin at 37° C. and 5% CO2. Knocking down p75NTR expression was performed according to manufacturer's instructions (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Briefly, MSCs were shifted to antibiotic-free medium over night when 60-80% confluent. Cells were transfected with Si-RNA against p75NTR receptor (sc-37268) or scrambled (sc-37007) with the aid of lipofectamine transfection reagent (sc-29528) for 6 h. DMEM medium containing 2× of FBS and antibiotic was added on top of pure transfection medium for an extra 12 h. Next, transfection medium was removed and cells were allowed to recover in 1×DMEM medium for 6 h. For in vivo studies, cells were collected in sterile PBS (50,000 cells/μL) for intravitreal injection 48 h after I/R. For additional set of experiments, MSCs were treated with the p75NTR modulator (LM11A-31, 200 nM). Confluent MSCs (80% confluent) were treated with LM11A-31 or its vehicle for 12 h. Then, condition media were collected as well as cell lysates using mirVANA™ PARIS™ Kit (Ambion Inc., Austin, TX, USA), according to manufacturer's instructions.
Mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg, Hospira, Inc., Lake Forest, IL, USA) and xylazine (10 mg/kg, Akorn, Decatur, IL, USA) mixture and complete anesthesia was confirmed by loss of reflex to sharp paw pinch. MSCs (100,000 cells/2 μL sterile PBS/eye) were injected intravitreally 48 h after I/R using a Hamilton syringe with a 33 gauge glass capillary.
3 or 7 days post intravitreal injection of MSCs, mice were euthanized in CO2 chamber (2% flow rate for 5 min), followed by cervical dislocation. Eyes were enucleated and fixed in 2% paraformaldehyde overnight. Retinas were dissected and permeabilized for 15 min with 0.3% Triton X-PBS then stained overnight at 4° C. with isolectin B4; biotinylated griffonia (bandeiraea) simplicifolia lectin I (GSL I, BSL I), (Vector Labs, Burlingame, CA, USA; 1% in 5% normal goat serum in 0.3% Triton X-PBS), followed by incubation with secondary antibody; Texas red® avidin D (Vector labs, Burlingame, CA, USA; 0.5% in 5% normal goat serum in 0.3% Triton X-PBS). Lectin-stained retinas were flat mounted onto Super-frost/Plus microscope slides (Fisher Scientific, Waltham, MA, USA) with the photoreceptor side facing down and imbedded in Vectashield mounting media for fluorescence (Vector Labs, Burlingame, CA, USA). Slides were photomicrographed at 40× using a Zeiss Axio Observer Z1 (Carl Zeiss Vision Inc., Thornwood, NY, USA).
The retinal vasculature was isolated as described previously [58]. Freshly enucleated eyes were fixed with 2% paraformaldehyde overnight. Retinal cups were dissected, washed in PBS, then incubated with 3% Difco-trypsin 250 (BD Biosciences, San Jose, CA, USA) in 25 mmol/l Tris buffer, pH 8, at 37° C. for 1-1.5 h. Vitreous and nonvascular cells were gently removed from the vasculature, which was soaked in several washes of 0.5% Triton X-100 to remove the neuronal retina. Trypsin-digested retinas were stained with periodic acid-Schiff and hematoxylin (PASH). Numbers of acellular capillaries were quantified in six different areas of the mid-retina using bright field microscopy (20×) in a masked manner by two different researchers. Acellular capillaries were identified as capillary-sized blood vessel tubes with no nuclei along their length.
All human retinal endothelial (HRE) cell studies were in accordance with Association for Research in Vision and Ophthalmology and Charlie Norwood Veterans Affairs Medical Center, research and ethics committee. HREs and supplies were purchased from Cell Systems Corporation (Kirkland, WA, USA) and VEC Technology Inc. (Rensselaer, NY, USA).
Retinas samples and MSCs lysates were processed using (mirVANA™ PARIS™ Kit, Ambion Inc., Austin, TX, USA) and RNA was purified and quantified as described by the manufacturer's instructions. A one-step quantitative RT-PCR kit (Invitrogen, Carlsbad, CA, USA) was used to amplify 10 ng retinal mRNA. Quantitative PCR was conducted using a StepOnePlus qPCR system (Applied BioSystems, Life Technologies, Waltham, MA, USA). For HREs, RNA isolation was performed using the TRIzol Reagent (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions. RNA concentration was quantified by spectrophotometry at 260 nm using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). A total of 1 μg of RNA was reverse transcribed to prepare cDNA using the RNA to cDNA EcoDry premix (Takara Bio USA Inc., Mountain View, CA, USA). Real-time PCR was performed using the CFX96 PCR instrument with matched primers (See Table 1) and Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). The following PCR parameters were used: initial denaturation cycle at 95° C. for 3 min, followed by 40 amplification cycles at 95° C. for 10 s, 56° C. for 15 s, and 72° C. for 1 min. The results are presented as the fold change in relative gene expression quantified using the delta-delta CT method and normalized to internal controls (18 S, GAPDH) and expressed relative to controls.
HREs were grown to confluence and then were wound with a single sterile cell scraper of constant diameter. Cells were divided into three groups, treated with regular media without FBS, and 1:1 mixture of FBS-free regular media and CM of vehicle- or treated-MSCs. Images of wounded areas were taken immediately after adding the treatment using a phase contrast microscope, and baseline were marked under the cell culture dish with blue marker. After 12 h, images of same field were taken and % cell migration was calculated. Each condition was verified in triplicate and was repeated using independent cultures.
Corning Matrigel Matrix (10 mg protein/mL) Growth Factor Reduced (Corning Inc., Cat. No. 354230, Corning, NY, USA) was used for tube formation assay. Then, 289 μL of cold (4° C.) Matrigel per well was added into chilled 24-well flat-bottom tissue culture plate (USA Scientific Inc., Ocala, FL, USA) and polymerized for 1 h at 37° C., 5% CO2 incubator. HREs were trypsinized and separated into different groups. Each group of cells was centrifuged and cell pellets were resuspended in regular media without FBS, same media with 1:1 mixture of FBS free regular media and CM of vehicle- or treated-MSCs. Then 300 μL of the cell suspension was added onto each well of Matrigel coated 24-well culture plate and incubated at 37° C. After 24-h, cells forming tube structures were analyzed by microscopy. Cells in five replicate fields of triplicate wells were digitally photographed with an EVOS microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA). The images for Tube formation assay were analyzed using Angiogenesis analyzer for imageJ. An area of 1.8 mm2 from each well was analyzed with imageJ ver. 1.53c, and 3 wells were analyzed per group. The software automatically generates the skeletonized trees overlay to highlight junction, branches, and segments (
Visual acuity was assessed behaviorally by training and testing mice on the “cue” version of the Morris water maze task. Mice used for this experiment underwent I/R in both eyes. They were trained for 3 days before I/R and visual acuity was tested at 3, 6, 9, 12 and 15 days post I/R. Animals were placed for 10 s on a platform in a tank of opaque water, 22-25° C., which was elevated above the water surface (2 cm) and clearly visible from any location in the tank. Subsequently, there were four trials per day for 3 days or until a stable performance plateau was reached. On each trial, animals started from different locations at the periphery of the tank and were allowed to swim to the escape platform. If they did not reach the platform in 60 s, they were gently guided to it by the investigator. They remained on the platform for 10 s. Visual impairment was diagnosed by higher escape time. Treatment groups received oral gavage of LM11A-31 (50 mg/kg/day) every other day, starting 48 h post I/R.
All the data are expressed as mean±SEM. Differences between 2 groups were detected using unpaired Student T-test. One-way ANOVA was used to assess significant differences between 3 groups. Two-way ANOVA was used to assess interaction between two variables: 2 genes (WT vs. p75NTR−/−) X I/R exposure (I/R vs Sham). Tukey-Kramer post-multiple comparisons was used for significant interactions among various groups. Significance for all tests was determined at α=0.05, Graphpad Prism, Ver.6.
Akt: Protein Kinase B; Bax: Bcl-2-like protein 4; Bcl-2B-cell lymphoma 2; CM: Conditioned medium; CXCR-4: Chemokine receptor type 4; CXCR-7: Chemokine receptor type 7; HREs Human retinal endothelial cells; I/R Ischemia reperfusion; MSCs Mesenchymal stem cells; NGF: Nerve growth factor; PASH: Periodic acid-Schiff and hematoxylin; P75NTR: p75 neurotrophin receptor; PDR: Proliferative diabetic retinopathy; SDF-1: Stromal cell-derived factor-1; VEGF: Vascular endothelial growth factor
Diabetes affects the whole body by causing a state of mild inflammation that can slow down the rolling of white blood cells to block or damage the small capillaries especially in the eye, kidney and the limbs. Leakage or death of the small capillaries leads to accumulation of fluids and limits the ability to regenerate after exposure to ischemic limb. Leakage of fluids in front of the macula disturb in visual acuity and results in appearance of protein the urine of diabetic patient. Current therapeutic options are limited for early stages they do not treat the whole body. Therefore, there is a great need to identify novel therapeutic targets that can improve the inflammation of the whole body and preserve both capillaries and neurons in the eye, kidney and the limbs. Translational studies showed the benefit of a new drug, LM11A-31 that can prevent the inflammation in the whole body and in particular the eye and kidney after the onset of diabetes. The studies quantified the benefit of the new drug on improving the damage of the small capillaries and fluid accumulation especially in the eye as well as visual function in ischemic and diabetic animals.
Diabetes and its microvascular complications are perceived as systemic low-grade inflammation manifested by positive association of circulating markers of inflammation. Nevertheless, several systemic orally administered drugs failed repeatedly in clinical trials. Hence, there is a need to identify viable targets that are “upstream” of the terminal effectors involved in the disease. The efficacy of a small p75NTR inhibitor, an orally bioavailable and safe drug (LM11A-31), has been used in an intervention using clinically relevant endpoints. The studies provide strong rationale for systemic targeting of the proinflammatory action of p75NTR and that it has proven efficacious in preventing diabetes-associated microvascular injury in retina, kidney and ischemic limbs.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.
This application is a Continuation of U.S. Non-Provisional patent application Ser. No. 18/060,160, filed on Nov. 30, 2022, which claims the benefit of U.S. Provisional Patent Application No. 63/284,489, filed on Nov. 30, 2021, each of which is incorporated by reference herein in its entirety.
This invention was made with government support under Grant Number R01-EY-022408 awarded by the National Institutes of Health. The government has certain rights in this invention.
Number | Date | Country | |
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63284489 | Nov 2021 | US |
Number | Date | Country | |
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Parent | 18060160 | Nov 2022 | US |
Child | 18491249 | US |