Patent Application
20240016804
This document relates to methods and materials for assessing and/or treating hypertensive disorders of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. For example, methods and materials provided herein can be used to determine if a female mammal has, or is likely to develop, a hypertensive disorder of pregnancy. In some cases, one or more senotherapeutic agents can be administered to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) to treat the female mammal.
Hypertensive disorders of pregnancy, including preeclampsia, remain one of the leading causes of maternal and fetal morbidity and mortality worldwide (Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy, Am. J. Obstet. Gynecol., 183(1):S1-522 (2000)). For example, preeclampsia (PE), a pregnancy-specific disorder characterized by hypertension and, commonly, proteinuria, commonly occurs after 20 weeks of gestation, and can affect approximately 5% of all pregnancies (Shohara et al., Cytotherapy, 14(10):1171-81 (2012)). Preeclampsia is frequently treated with blood pressure treatment, and premature delivery or termination of pregnancy is often recommended.
Preeclampsia can be difficult to diagnose, and there is no specific treatment of preeclampsia or its related health complications that occur later in life. This document provides methods and materials related to assessing and/or treating hypertensive disorders of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. In some cases, this document provides methods and materials for identifying a female mammal as having, or as being likely to develop, a hypertensive disorder of pregnancy. For example, this document provides methods and materials for detecting the presence or absence of an elevated level of expression of one or more senescence-associated secretory phenotype (SASP) polypeptides within a female mammal (e.g., a female human) and classifying the female mammal as having, or as being likely to develop, a hypertensive disorder of pregnancy if the presence of an elevated level of expression of one or more SASP factor polypeptides is detected. In some cases, this document provides methods and materials for treating a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. For example, this document provides methods and materials for using one or more senotherapeutic agents to treat a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy.
As described herein, the presence of an elevated level of expression of one or more SASP polypeptides in a sample obtained from a female mammal can be used to identify that female mammal as having, or as being at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. For example, mesenchymal stem cells (MSCs) isolated from adipose tissue of women having preeclampsia can have a lower percentage of live MSCs as compared to MSCs isolated from adipose tissue of women having a normotensive pregnancy, and the live MSCs can exhibit a senescent phenotype, upregulation of senescence markers and SASP components, and lower angiogenic potential. As also described herein, one or more senotherapeutic agents such as dasatinib can be used to treat a hypertensive disorder of pregnancy. For example, treatment of MSCs isolated from adipose tissue of women having preeclampsia with dasatinib can increase the number of apoptotic MSCs and decreased expression of p16 and six SASP components as compared to MSCs isolated from adipose tissue of women having a normotensive pregnancy.
Having the ability to identify a female mammal as having or as being at risk of developing preeclampsia and/or having the ability to delay or prevent the development of a hypertensive disorder of pregnancy such as preeclampsia provides a unique and unrealized opportunity to allow women to carry a pregnancy to term safely, with fewer small-for-gestational age infants, and fewer stillbirths. In some cases, a non-pregnant woman at risk of developing a hypertensive disorder of pregnancy (e.g., preeclampsia) can be administered one or more senotherapeutic agents to prevent the development of such a disorder during pregnancy. For example, a non-pregnant woman who has had one or more previous unsuccessful pregnancies due to a hypertensive disorder of pregnancy (e.g., preeclampsia) can be administered one or more senotherapeutic agents prior to becoming pregnant to prevent the development of preeclampsia in a next pregnancy.
In general, one aspect of this document features methods for assessing a female mammal for a hypertensive disorder of pregnancy or the likelihood of developing the hypertensive disorder of pregnancy. The methods can include, or consist essentially of, (a) detecting the presence or absence of an elevated level of expression of a SASP polypeptide in a sample from the mammal; (b) classifying the mammal as having, or as likely to develop, the hypertensive disorder of pregnancy based at least in part on the presence of the elevated level, and (c) classifying the mammal as lacking, or as unlikely to develop, the hypertensive disorder of pregnancy based at least in part on the absence of the elevated level. The mammal can be a human. The mammal can be not pregnant. The mammal can be pregnant. The hypertensive disorder of pregnancy can be preeclampsia. The SASP polypeptide can be interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1), osteopontin, activine A, eutaxin, GDF15, IL1 alpha, IL1 beta, MIPa, Rantese, MMP2 miRNA, MMP3 miRNA, MMP9 miRNA, or MMP12 miRNA. The sample can be whole blood, serum, plasma, peripheral blood mononucleated cells (PBMCs), urine, cerebrospinal fluid (CSF), adipose tissue, or skin tissue. In another aspect, this document features methods for treating a non-pregnant female mammal having a hypertensive disorder of pregnancy or likely to develop the hypertensive disorder of pregnancy. The methods can include, or consist essentially of, (a) detecting an elevated level of expression of a SASP polypeptide in a sample obtained from a mammal; and (b) administering a senolytic agent to the mammal. The mammal can be a human. The hypertensive disorder of pregnancy can be preeclampsia. The senolytic agent can be dasatinib. The SASP polypeptide can be IL-6, IL-8, MCP-1, PAI-1, osteopontin, activine A, eutaxin, GDF15, IL1 alpha, IL1 beta, MIPa, Rantese, MMP2 miRNA, MMP3 miRNA, MMP9 miRNA, or MMP12 miRNA. The sample can be whole blood, serum, plasma, PBMCs, urine, CSF, adipose tissue, or skin tissue.
In another aspect, this document features methods for treating a hypertensive disorder of pregnancy. The methods can include, or consist essentially of, administering a senolytic treatment to a non-pregnant female mammal identified as having an elevated level of expression of a SASP polypeptide in a sample obtained from the mammal. The mammal can be a human. The hypertensive disorder of pregnancy can be preeclampsia. The senolytic agent can be dasatinib. The SASP polypeptide can be IL-6, IL-8, MCP-1, PAI-1, osteopontin, activine A, eutaxin, GDF15, IL1 alpha, IL1 beta, MIPa, Rantese, MMP2 miRNA, MMP3 miRNA, MMP9 miRNA, or MMP12 miRNA. The sample can be whole blood, serum, plasma, PBMCs, urine, CSF, adipose tissue, or skin tissue.
In another aspect, this document features methods for delaying the onset of a comorbidity of a hypertensive disorder of pregnancy. The methods can include, or consist essentially of, administering a senolytic treatment to a non-pregnant female mammal (a) having had a pregnancy affected by a hypertensive disorder of pregnancy, and (b) identified as having an elevated level of expression of a SASP polypeptide in a sample obtained from the mammal. The mammal can be a human. The hypertensive disorder of pregnancy can be preeclampsia. The senolytic agent can be dasatinib. The comorbidity of the hypertensive disorder of pregnancy can be diabetes mellitus type 2, cardiovascular disease, stroke, kidney disease, vascular dysfunction, atherosclerotic burden, fat inflammation, vascular injury, cardiac arrhythmias, congestive heart failure, dementia, depression, substance abuse, hyperlipidemia, arthritis, cancer, asthma, chronic obstructive pulmonary disease, or osteoporosis. The SASP polypeptide can be IL-6, IL-8, MCP-1, PAI-1, osteopontin, activine A, eutaxin, GDF15, IL1 alpha, IL1 beta, MIPa, Rantese, MMP2 miRNA, MMP3 miRNA, MMP9 miRNA, or MMP12 miRNA. The sample can be whole blood, serum, plasma, PBMCs, urine, CSF, adipose tissue, or skin tissue.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials for identifying and/or treating female mammals (e.g., female humans) having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. In some cases, this document provides methods and materials for identifying a female mammal as having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. As described herein, an elevated level of expression of one or more SASP polypeptides can be present in a sample obtained from a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. In some cases, this document provides methods and materials for identifying a female mammal as having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. For example, a female mammal (e.g., a female human) can be identified as having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy based, at least in part, on the presence of an elevated level of expression of one or more SASP polypeptides in a sample obtained from the female mammal. In some cases, this document provides methods and materials for treating a female mammal (e.g., a female human) having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. For example, a female mammal identified as having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy as described herein (e.g., based, at least in part, on the presence of an elevated level of expression of one or more SASP polypeptides in a sample from the female mammal) can be administered one or more senotherapeutic agents (e.g., dasatinib) to treat the female mammal. In some cases, one or more senotherapeutic agents can be used to treat a female mammal having had a previous pregnancy affected by a hypertensive disorder of pregnancy (e.g., prior to a subsequent pregnancy to prevent or delay developing a hypertensive disorder of pregnancy in that subsequent pregnancy).
In some cases, the presence of an elevated level of expression of one or more (e.g., one, two, three, four, or more) SASP polypeptides in a sample (e.g., a sample obtained from a female mammal such as a female human) can be used to identify the female mammal as having, or as being likely to develop, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. The term “elevated level” as used herein with respect to a level of expression of one or more SASP polypeptides refers to any level that is greater than a reference level of expression of a SASP polypeptide. The term “reference level” as used herein with respect to expression of a SASP polypeptide refers to the level of expression of the SASP polypeptide typically observed in a sample (e.g., a control sample) from one or more comparable female mammals (e.g., female humans of comparable age) that do not have a to hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. Control samples can include, without limitation, samples from female mammals having a normotensive pregnancy, and samples from non-pregnant female mammals. In some cases, an elevated level of expression of a SASP polypeptide can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater than a reference level of expression of the SASP polypeptide. In some cases, an elevated level of expression of a SASP polypeptide can be a level that is greater than 1 picogram (pg) per μL. In some cases, when control samples have an undetectable level of expression of a SASP polypeptide, an elevated level can be any detectable level of expression of the SASP polypeptide. It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is an elevated level. In some cases, an elevated level of expression of a SASP polypeptide for a sample can be a level where greater than 0.0001 percent of the cells of the sample express detectable levels of expression of SASP polypeptides. For example, when greater than 0.0001 percent of the cells of a sample are determined to express a detectable level of expression of a SASP polypeptide, then that sample can be classified as having an elevated level of expression of the SASP polypeptide.
The presence of an elevated level of expression of any appropriate SASP polypeptide (e.g., in a sample such as a sample obtained from a female mammal such as a female human) can be used to identify a female mammal as having, or as being likely to develop, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. Examples of SASP polypeptides include, without limitation, IL-6, IL-8, MCP-1, PAI-1, osteopontin, activine A, eutaxin, GDF15, IL1 alpha, IL1 beta, MIPa, Rantese, MMP2 miRNA, MMP3 miRNA, MMP9 miRNA, and MMP12 miRNA. Exemplary polypeptide sequences (and the nucleic acids encoding such polypeptides) of SASP polypeptides can be as set forth in the National Center for Biotechnology Information (NCBI) databases at, for example, Accession No. NM 001318095.1, Accession No. NM_002982, and Accession No. NM_001165413.
Any appropriate mammal can be assessed and/or treated as described herein. In some cases, a female mammal can be a pregnant mammal. For example, when a senotherapeutic agent is senomorphic agent, a female mammal can be a pregnant mammal. In some cases, a female mammal can be a non-pregnant mammal. For example, when a senotherapeutic agent is senolytic agent, a female mammal can be a non-pregnant mammal. In some cases, a female mammal can have experienced one or more previous pregnancies (e.g., one or more previous pregnancies affected by a hypertensive disorder of pregnancy). In some cases, a female mammal can be an obese female mammal (e.g., a female mammal that is overweight). Examples of female mammals that can be treated using a composition containing one or more senotherapeutic agents as described herein include, without limitation, humans, non-human primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, mice, and rats. In some cases, a composition containing one or more senotherapeutic agents can be administered to a human having a hypertensive disorder of pregnancy (e.g., preeclampsia) to treat the human. In some cases, a composition containing one or more senotherapeutic agents can be administered to a female human at risk of developing one or more comorbidities associated with a hypertensive disorder of pregnancy to slow the onset or progression of the one or more comorbidities associated with a hypertensive disorder of pregnancy within the human.
Any appropriate sample from a female mammal (e.g., a female human) can be assessed as described herein (e.g., for the presence, absence, or level of expression of one or more SASP polypeptides). In some cases, a sample can be a biological sample. In some cases, a sample can contain one or more biological molecules (e.g., nucleic acids such as DNA and RNA, polypeptides, carbohydrates, lipids, hormones, and/or metabolites). Examples of samples that can be assessed as described herein include, without limitation, fluid samples (e.g., whole blood, serum, plasma, PBMCs, urine, and CSF), and tissue samples (e.g., adipose tissue, and skin tissue). A biological sample can be a fresh sample or a fixed sample (e.g., a formaldehyde-fixed sample or a formalin-fixed sample). In some cases, a biological sample can be a processed sample (e.g., an embedded sample such as a paraffin or OCT embedded sample, or processed to isolate or extract one or more biological molecules). For example, a blood (e.g., serum) sample can be obtained from a female mammal and can be assessed for the presence, absence, or level of expression of one or more SASP polypeptides to determine if the mammal has, or is likely to develop, a hypertensive disorder of pregnancy such as preeclampsia based, at least in part, on the presence of an elevated level of expression of one or more SASP polypeptides in the sample.
Any appropriate method can be used to detect the presence, absence, or level of expression of one or more SASP polypeptides within a sample (e.g., a sample obtained from a female mammal such as a female human). In some cases, the presence, absence, or level of expression of one or more SASP polypeptides within a sample can be determined by detecting the presence, absence, or level of one or more SASP polypeptides in the sample. For example, immunoassays (e.g., immunohistochemistry (IHC) techniques, and western blotting techniques), mass spectrometry techniques (e.g., proteomics-based mass spectrometry assays or targeted quantification-based mass spectrometry assays), and enzyme-linked immunosorbent assays (ELISAs) can be used to determine the presence, absence, or level of one or more SASP polypeptides in a sample. When an immunoassay is used to determine the presence, absence, or level of one or more SASP polypeptides in a sample, the immunoassay can use any appropriate antibody. Examples of antibodies that can be used in an immunoassay to determine the presence, absence, or level of one or more SASP polypeptides in a sample include, without limitation, human activin A antibodies (e.g., R&D systems DAC008), human total MMP-7 antibodies (e.g., R&D systems DMP700), human IL-6 antibodies (e.g., R&D systems
D6050), human serpin E1/PAI-1 antibodies (e.g., R&D systems DSE100), human CCL2/MCP-1 antibodies (e.g., R&D systems DCP00), human osteopontin antibodies (e.g., eBioscience BMS2066), and human MMP-1 antibodies (e.g., RayBiotech., Inc. ELH-MMP-1).
In some cases, the presence, absence, or level of expression of one or more SASP polypeptides within a sample can be determined by detecting the presence, absence, or level of mRNA encoding a SASP polypeptide in the sample. For example, polymerase chain reaction (PCR)-based techniques such as quantitative reverse transcription (RT)-PCR (qPCR) techniques, RNA in situ hybridization (ISH), and RNA sequencing can be used to determine the presence, absence, or level of mRNA encoding a SASP polypeptide in the sample. In some cases, the presence, absence, or level of expression of one or more SASP polypeptides within a sample can be determined by qPCR. In some cases, the presence, absence, or level of expression of one or more SASP polypeptides within a sample can be determined as described in Example 3.
In some cases, when a female mammal (e.g., a female human) is identified as having, or as being likely to develop, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy based, at least in part, on an elevated level of expression of one or more SASP polypeptides in a sample as described herein, the presence of a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy can be confirmed using one or more diagnostic techniques. Any appropriate technique can be used to confirm that a female mammal has, or is at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. For example, proteinuria, a low platelet count, impaired liver function, signs of kidney problems (e.g., other than proteinuria), pulmonary edema, new-onset headaches or visual disturbances, a blood pressure in excess of 140/90 mm Hg and/or seizures after the 20th week of pregnancy can be used to confirm a female mammal as having a hypertensive disorder of pregnancy. For example, a history of preeclampsia (e.g., during a previous pregnancy), chronic hypertension, first pregnancy, new paternity (e.g., as compared to a previous pregnancy), age (e.g., pregnancy in women younger than 20 and/or pregnancy in women older than 40), obesity, pregnancy with multiples (e.g., twins, triplets, or other multiples), interval between pregnancies (e.g., pregnancies less than two years or more than 10 years apart), history of certain medical conditions (e.g., chronic high blood pressure, migraines, type 1 or type 2 diabetes, and/or kidney disease) prior to pregnancy, and/or conception by in vitro fertilization can be used to confirm a female mammal as being at risk of developing a hypertensive disorder of pregnancy. For example, a female mammal having had a hypertensive disorder of pregnancy (e.g., preeclampsia) during one or more previous pregnancies can be confirmed as being at risk of developing one or more comorbidities associated with a hypertensive disorder of pregnancy.
When assessing and/or treating a hypertensive disorder of pregnancy as described herein, the hypertensive disorder of pregnancy can be any type of hypertensive disorder of pregnancy. Examples of hypertensive disorders of pregnancy that can be treated as described herein include, without limitation, preeclampsia, postpartum preeclampsia, eclampsia, chronic hypertension, preeclampsia superimposed on chronic hypertension, and gestational hypertension (e.g., transient hypertension of pregnancy or chronic hypertension identified in the latter half of pregnancy).
When assessing and/or treating a comorbidity associated with a hypertensive disorder of pregnancy as described herein, the comorbidity associated with a hypertensive disorder of pregnancy can be any type of comorbidity associated with a hypertensive disorder of pregnancy. In some cases, a comorbidity can be an age-related disease. In some cases, a comorbidity associated with a hypertensive disorder of pregnancy can be present at the same time as a hypertensive disorder of pregnancy (e.g., during the pregnancy). In some cases, a comorbidity associated with a hypertensive disorder of pregnancy can develop after the pregnancy. Examples of comorbidities (e.g., future comorbidities such as one or more comorbidities that can develop following the completion of the female mammal's pregnancy) associated with a hypertensive disorder of pregnancy that can be treated as described herein include, without limitation, hypertension, diabetes (e.g., diabetes mellitus type 2), vascular disease (e.g., cardiovascular disease (CVD) such as coronary artery disease), stroke, kidney disease (e.g., chronic kidney disease), vascular dysfunction, atherosclerotic burden, inflammation (e.g., fat inflammation and/or systemic inflammation), vascular injury, cardiac arrhythmias, congestive heart failure, dementia, and hyperlipidemia.
Once identified as having, or as being at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy, a female mammal can be administered or instructed to self-administer one or more (e.g., one, two, three, four, five or more) senotherapeutic agents. Any appropriate senotherapeutic agent can be used as described herein. A senotherapeutic agent that can be used as described herein can be any type of molecule (e.g., small molecules or polypeptides). In some cases, a senotherapeutic agent can be a senolytic agent (i.e., an agent having the ability to induce cell death in senescent cells). In some cases, a senotherapeutic agent can be a senomorphic agent (i.e., an agent having the ability to suppress senescent phenotypes without cell killing). Examples of senotherapeutic agents that can be used as described herein (e.g., to treat a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy such as preeclampsia and/or one or more comorbidities associated with a hypertensive disorder of pregnancy) can include, without limitation, dasatinib, quercetin, navitoclax, A1331852, A1155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin3a, ruxolitinib, metformin, and rapamycin.
One or more senotherapeutic agents (e.g., dasatinib) can be administered to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy at any appropriate time. In some cases, one or more senotherapeutic agents can be administered to a female mammal (e.g., a female human) in need thereof (e.g., a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy) as described herein prior to the female mammal becoming pregnant. In some cases, one or more senotherapeutic agents can be administered to a female mammal (e.g., a female human) in need thereof (e.g., a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy) as described herein during the female mammal's pregnancy. In some cases, one or more senotherapeutic agents can be administered to a female mammal (e.g., a female human) in need thereof (e.g., a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy) as described herein following the completion of the female mammal's pregnancy.
In some cases, a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy can be treated with one or more senotherapeutic agents (e.g., dasatinib) to alleviate (e.g., to reduce or eliminate) one or more (e.g., one, two, three, four, five or more) symptoms of the hypertensive disorder of pregnancy. A symptom of a hypertensive disorder of pregnancy can be any appropriate symptom. Examples of symptoms of preeclampsia include, without limitation, hypertension, proteinuria, headaches, blurred vision, temporary loss of vision, sensitivity to light, abdominal pain, nausea and/or vomiting, decreased urine output, thrombocytopenia, impaired liver function, shortness of breath (e.g., caused by fluid in the lungs), and edema (e.g., pulmonary edema). Each of these symptoms of a hypertensive disorder of pregnancy can be identified and/or monitored using clinical techniques as described elsewhere (see, e.g., Am. J. Obstet. Gynecol., 183(1):S1-S22 (2000)) For example, one or more senotherapeutic agents can be administered to a female mammal (e.g., a female human) in need thereof (e.g., a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy) as described herein to reduce the severity of one or more symptoms a hypertensive disorder of pregnancy in the female mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, a female mammal having, or at risk of developing, a comorbidity associated with a hypertensive disorder of pregnancy (e.g., a female mammal having had a pregnancy affected by a hypertensive disorder of pregnancy) can be treated with one or more senotherapeutic agents (e.g., dasatinib) to delay or prevent the onset of a comorbidity associated with a hypertensive disorder of pregnancy. For example, a female mammal (e.g., a female human) who has previously had one or more previous pregnancies affected by a hypertensive disorder of pregnancy can be administered one or more senotherapeutic agents to delay the onset of a comorbidity associated with a hypertensive disorder of pregnancy by about 7 to about 10 years (e.g., as compared to the age of development of the same comorbidity in a female mammal who experienced a comparable hypertensive disorder of pregnancy and was not administered the one or more senotherapeutic agents). For example, a female mammal (e.g., a female human) who has previously had one or more previous pregnancies affected by a hypertensive disorder of pregnancy can be administered one or more senotherapeutic agents to prevent the onset of a comorbidity associated with a hypertensive disorder of pregnancy (e.g., as compared to the age of development of the same comorbidity in a female mammal who experienced a comparable hypertensive disorder of pregnancy and was not administered the one or more senotherapeutic agents).
In some cases, a female mammal having, or at risk of developing, a comorbidity associated with a hypertensive disorder of pregnancy (e.g., a female mammal having had a pregnancy affected by a hypertensive disorder of pregnancy) can be treated with one or more senotherapeutic agents (e.g., dasatinib) to alleviate (e.g., to reduce or eliminate) one or more (e.g., one, two, three, four, five or more) symptoms of the comorbidity associated with a hypertensive disorder of pregnancy. A symptom of a comorbidity associated with a hypertensive disorder of pregnancy can be any appropriate symptom. Examples of symptoms of hypertension associated with a hypertensive disorder of pregnancy include, without limitation, headaches, chest pain, shortness of breath or nosebleeds, epigastric pain, dizziness, and palpitations. Each of these symptoms of hypertension associated with a hypertensive disorder of pregnancy can be identified and/or monitored using clinical techniques as described elsewhere (see, e.g., Am. J. Obstet. Gynecol., 183(1):S1-S22 (2000)). Examples of symptoms of diabetes (e.g., diabetes mellitus type 2) associated with a hypertensive disorder of pregnancy include, without limitation, increased thirst, frequent urination, increased hunger, unintended weight loss, fatigue, blurred vision, slow-healing sores, frequent infections, areas of darkened skin, and fainting spells (e.g., due to hypoglycemic episodes). Each of these symptoms of diabetes (e.g., diabetes mellitus type 2) associated with a hypertensive disorder of pregnancy can be identified and/or monitored using clinical techniques as described elsewhere (see, e.g., Weissgerber et al., Curr Diab Rep. 15(3):9 (2015)). Examples of symptoms of a CVD such as coronary artery disease associated with a hypertensive disorder of pregnancy include, without limitation, chest pain (angina), shortness of breath, heart attack, pulmonary edema, and cardiac arrhythmias. Each of these symptoms of coronary artery disease associated with a hypertensive disorder of pregnancy can be identified and/or monitored using clinical techniques as described elsewhere (see, e.g., Mosca et al., Circulation. 99(18):2480-4 (1999)). Examples of symptoms of stroke associated with a hypertensive disorder of pregnancy include, without limitation, trouble with speaking and understanding, paralysis or numbness (e.g., of the face, arm or leg), trouble with seeing in one or both eyes, headache, trouble with walking, trouble with swallowing, confusion, and loss of conciseness. Each of these symptoms of stroke associated with a hypertensive disorder of pregnancy can be identified and/or monitored using clinical techniques as described elsewhere (see, e.g., Bushnell et al., Stroke. 45(5):1545-88. (2014)). Examples of symptoms of kidney disease associated with a hypertensive disorder of pregnancy include, without limitation, nausea, vomiting, loss of appetite, fatigue and weakness, sleep problems, changes in amounts of urinate, decreased mental sharpness, muscle twitches and cramps, swelling of feet and ankles, persistent itching, chest pain (e.g., if fluid builds up around the lining of the heart), shortness of breath (e.g., if fluid builds up in the lungs), metallic taste, decreased appetite, and weight loss. Each of these symptoms of kidney disease associated with a hypertensive disorder of pregnancy can be identified and/or monitored using clinical techniques as described elsewhere (see, e.g., Kidney Disease: Improving Global Outcomes (KDIGO) CKD-MBD Work, Kidney Int Suppl, August 2009 (113):S1-S130 (2009)). For example, one or more senotherapeutic agents can be administered to a female mammal (e.g., a female human) in need thereof (e.g., a female mammal having, or at risk of developing, a comorbidity associated with a hypertensive disorder of pregnancy) as described herein to reduce the severity of one or more symptoms of a comorbidity associated with a hypertensive disorder of pregnancy in the female mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy can be treated with one or more senotherapeutic agents (e.g., dasatinib) to clear one or more senescent cells from within the female mammal. A senescent cell can be any type of cell. Examples of senescent cells that can be cleared as described herein include, without limitation, preadipocytes, endothelial cells, dendritic cells, fibroblasts, astrocytes, myofibroblast, pancreatic beta cells, and osteoblasts. A senescent cell can be cleared from any location within the female mammal. Examples of locations from which a senescent cell can be cleared include, without limitation, adipose tissue, vascular system, bone, marrow, brain, heart, pancreas, liver, kidney, muscle, and blood of the female mammal. In some cases, administering one or more senotherapeutic agents to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy can be effective to clear one or more senescent cells from adipose tissue within the female mammal. For example, one or more senotherapeutic agents can be administered to a female mammal (e.g., a female human) in need thereof (e.g., a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or a comorbidity associated with a hypertensive disorder of pregnancy) as described herein to clear, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent of the senescent cells present from a location (e.g., adipose tissue) within the female mammal.
In some cases, a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy can be treated with one or more senotherapeutic agents (e.g., dasatinib) to increase (e.g., restore) angiogenesis (e.g., to increase the angiogenic potential of a cell) within the female mammal. In some cases, a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy can be treated with a composition including one or more senotherapeutic agents (e.g., dasatinib) to alleviate (e.g., to reduce or eliminate) age-related impairment of angiogenesis in the female mammal. Angiogenesis can be increased in any appropriate type of cell. In some cases, angiogenesis can be increased in a stem cell. Examples of cells in which angiogenesis can be increased as described herein include, without limitation, MSCs (e.g., an adipose-derived MSC), and endothelial cells. For example, clearance of one or more senescent cells from a population of cells including MSCs and/or endothelial cells can increase angiogenesis of the MSCs and/or the endothelial cells. In some cases, administering one or more senotherapeutic agents to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive to disorder of pregnancy can be effective to increase angiogenesis in one or more MSCs within the female mammal. For example, one or more senotherapeutic agents can be administered to a female mammal (e.g., a female human) in need thereof (e.g., a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or a comorbidity associated with a hypertensive disorder of pregnancy) as described herein to increase angiogenesis in, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent of cells (e.g., MSCs) present within the female mammal.
In some cases, a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy can be treated with one or more senotherapeutic agents (e.g., dasatinib) to alleviate (e.g., to reduce or eliminate) inflammation in the female mammal. A level (e.g., a systemic level) of any appropriate inflammatory factor (e.g., cytokines, chemokines, and matrix proteases) can be altered (e.g., increased or decreased) to alleviate inflammation in a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. In cases where an inflammatory factor is a pro-inflammatory factor (e.g., SASP polypeptides such as IL-6, IL-8, MCP-1, PAI-1, GDF15, MIPa, eutaxin, osteopontin, and activine A), the pro-inflammatory factor can be decreased. In cases where an inflammatory factor is an anti-inflammatory factor, the anti-inflammatory factor can be increased. For example, one or more senotherapeutic agents can be administered to a female mammal (e.g., a female human) in need thereof (e.g., a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or a comorbidity associated with a hypertensive disorder of pregnancy) as described herein to alter a level of an inflammatory factor in a cell within the female mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. Inflammation at any appropriate location within the female mammal can be alleviated. Examples of locations from which inflammation can be alleviated as described herein include, without limitation, adipose tissue, brain, pancreas, liver, kidney, vasculature, blood, brain, and CSF. In some cases, administering one or more senotherapeutic agents to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy can be effective to alleviate adipose inflammation within the female mammal.
In some cases, one or more senotherapeutic agents (e.g., dasatinib) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. For example, one or more senotherapeutic agents can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and corn oil.
A composition including one or more senotherapeutic agents (e.g., dasatinib) can be designed for any type of administration to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. For example, a composition including one or more senotherapeutic agents can be designed for oral or parenteral (including, without limitation, a subcutaneous, intramuscular, intravenous, intradermal, intra-cerebral, intrathecal, or intraperitoneal (i.p.) injection) administration to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. In some cases, compositions suitable for oral administration can be in the form of a food supplement. In some cases, compositions suitable for oral administration can be in the form of a drink supplement. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
A composition including one or more senotherapeutic agents (e.g., dasatinib) can be administered to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy in any appropriate amount (e.g., any appropriate dose). Effective amounts can vary depending on the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician. An effective amount of a composition containing one or more senotherapeutic agents can be any amount that can treat a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy as described herein without producing significant toxicity to the female mammal. For example, an effective amount of dasatinib can be from about 1 micromolar (μM) to about 20 μM (e.g., from about 1 μM to about 15 μM, from about 1 μM to about 12 μM, from about 1 μM to about 10 μM, from about 1 μM to about 7 μM, from about 1 μM to about 5 μM, from about 5 μM to about 20 μM, from about 8 μM to about 20 μM, from about 10 μM to about 20 μM, from about 12 μM to about 20 μM, from about 15 μM to about 20 μM, from about 5 μM to about 15 μM, from about 8 μM to about 12 μM, from about 5 μM to about 10 μM, or from about 10 μM to about 15 μM). For example, an effective amount of dasatinib can be from about 1 milligrams per kilogram body weight (mg/kg) to about 20 mg/kg (e.g., about 5 mg/kg). The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the female mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and/or severity of the hypertensive disorder of pregnancy (e.g., preeclampsia) and/or the one or more comorbidities associated with a hypertensive disorder of pregnancy in the female mammal being treated may require an increase or decrease in the actual effective amount administered.
A composition containing one or more senotherapeutic agents (e.g., dasatinib) can be administered to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy in any appropriate frequency. The frequency of administration can be any frequency that can treat a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy without producing significant toxicity to the female mammal. For example, the frequency of administration can be from about once a day to about once a week, from about once a week to about once a month, or from about twice a month to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and/or route of administration may require an increase or decrease in administration frequency.
A composition containing one or more senotherapeutic agents (e.g., dasatinib) can be administered to a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy for any appropriate duration. An effective duration for administering or using a composition containing one or more senotherapeutic agents can be any duration that can treat a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy without producing significant toxicity to the female mammal. For example, the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. In some cases, the effective duration can range in duration from about 10 years to about a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and/or route of administration.
In some cases, a composition containing one or more (e.g., one, two, three, four, five or more) senotherapeutic agents (e.g., dasatinib) can include the one or more senotherapeutic agent(s) as the sole active ingredient(s) in the composition effective to treat a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy.
In some cases, a composition containing one or more (e.g., one, two, three, four, five or more) senotherapeutic agents (e.g., dasatinib) can include one or more (e.g., one, two, three, four, five or more) additional active agents (e.g., therapeutic agents) in the composition that are effective to treat a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy.
In some cases, a female mammal having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy being treated as described herein (e.g., by administering one or more senotherapeutic agents such as dasatinib) also can be treated with one or more (e.g., one, two, three, four, five or more) additional therapeutic agents. A therapeutic agent used in combination with one or more senotherapeutic agents described herein can be any appropriate therapeutic agent. Examples of therapeutic agents that can be used in combination with one or more senotherapeutic agents described herein include, without limitation, antihypertensives, corticosteroids, anticonvulsant medications, aspirin (e.g., low-dose aspirin), calcium supplements, and magnesium sulfate. In some cases, the one or more additional therapeutic agents can be administered together with the one or more senotherapeutic agents (e.g., in a composition containing one or more senotherapeutic agents and containing one or more additional therapeutic agents). In some cases, the one or more (e.g., one, two, three, four, five or more) additional therapeutic agents can be administered independent of the one or more senotherapeutic agents. When the one or more additional therapeutic agents are administered independent of the one or more senotherapeutic agents, the one or more senotherapeutic agents can be administered first, and the one or more additional therapeutic agents administered second, or vice versa.
In some cases, methods for treating a female mammal (e.g., a female human) having, or at risk of developing, a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy as described herein (e.g., by administering one or more senotherapeutic agents such as dasatinib) also can include subjecting the mammal to one or more (e.g., one, two, three, four, five or more) additional treatments (e.g., therapeutic interventions) that are effective to treat a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy to treat the mammal. An example of an additional treatment that can be used as described herein to treat a hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy include, without limitation, stem cell (e.g., MSC) transplantation (e.g., autologous stem cell transplantation). In some cases, MSCs present in an autologous stem cell transplantation can be treated with one or more senotherapeutic agents. For example, when a female mammal identified as having, or at risk of developing, a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy is a pregnant female mammal, the pregnant female mammal can be treated by providing the pregnant female mammal with an autologous MSC transplantation where the MSCs have been treated with one or more senotherapeutic agents. In some cases, the one or more additional treatments that are effective to treat one or more symptoms of a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy can be performed at the same time as the administration of the one or more senotherapeutic agents. In some cases, the one or more additional treatments that are effective to treat one or more symptoms of a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy can be performed before and/or after the administration of the one or more senotherapeutic agents.
In some cases, methods for treating a female mammal (e.g., a female human) having, or at risk of developing, hypertensive disorder of pregnancy (e.g., preeclampsia) and/or one or more comorbidities associated with a hypertensive disorder of pregnancy as described herein (e.g., by administering one or more senotherapeutic agents) also can include monitoring the female mammal being treated. Any appropriate method can be used to monitor the severity of a hypertensive disorder of pregnancy and/or one or more comorbidities associated with a hypertensive disorder of pregnancy in a female mammal. In some cases, methods described herein also can include monitoring a female mammal being treated as described herein for toxicity. The level of toxicity, if any, can be determined by assessing a female mammal's clinical signs and symptoms before and after administering a known amount of a particular composition. It is noted that the effective amount of a particular composition administered to a female mammal can be adjusted according to a desired outcome as well as the female mammal's response and level of toxicity.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
This Example demonstrates that accelerated aging, in general, and cellular senescence, a cellular program characterized by irreversible proliferation arrest in particular, play a mechanistic role in the pathophysiology of preeclampsia (PE).
Mean epigenetic age (using a validated “epigenetic clock” method) was 2.4 years higher in women with preeclampsia (PE) vs. normotensive pregnancy (NP) at delivery (
Cellular senescence and a senescence-associated secretory phenotype (SASP), a distinctive secretome consisting of inflammatory cytokines and reactive oxygen species that ultimately leads to vascular injury were also examined. MSCs, multipotent cells with immunomodulatory, anti-inflammatory, and pro-angiogenic activities, which were isolated from the abdominal fat tissue (3-5 g) removed during C-sections from normotensive and PE pregnancies were studied. An abundance of cells stained positively for senescence-associated B-galactosidase (SABG) cultured MSC from PE, but not in cultured MSC from NP. Treatment with senolytics significantly reduced the senescence cell number in PE (
These results demonstrate that senolytics can be used as a targeted treatment for PE.
The Rochester Epidemiology Project (REP) medical record-linkage system was used to establish a cohort consisting of all women (n=8,177) who were residents of Olmsted County, Minnesota and delivered (liveborn or stillborn, ≥20 weeks' gestation) between Jan. 1, 1976 and Dec. 31, 1982 (
Women who either did not consent to the use of their medical records for research or with insufficient pregnancy information reported in the medical record were excluded. A pregnancy was classified as having sufficient information to determine HDP status if there was at least one blood pressure measurement from a prenatal visit and at least one blood pressure measurement from admission for delivery. The final cohort used for estimating the per-pregnancy incidence of HDP consisted of 7,544 mothers with 9,862 pregnancies. For the incidence of HDP per-woman, a sub-cohort of 1,839 women was identified. These women had their first deliveries between1976 and 1982 while residents of Olmsted County and who were also residents of Olmsted County by the end of their childbearing years, and for whom sufficient information was reported for all of their pregnancies.
Women with diagnostic codes indicative of a possible HDP occurring between Jan. 1, 1976 and Dec. 31, 1982, were first identified and their charts were fully abstracted (a list of codes is shown in Table 1). Every chart of each remaining woman in the cohort without a code suggestive of a possible HDP was then screened. A positive screen was defined as two elevated blood pressures, either systolic blood pressures (SBP)>140 mmHg and/or diastolic blood pressures (DBP)>90 mmHg at any prenatal visit, during admission for delivery, or postnatally before leaving the hospital. All screen positive charts were then fully abstracted. Screen-negative charts were categorized as normotensive pregnancies.
From the first prenatal visit through 12 weeks post-partum, all data regarding blood pressures, dipstick protein, and hypertensive medication use at each visit were recorded and dated. The following laboratory values were collected between the first prenatal visit and up to 72 hours post-partum: 24-hour protein, serum creatinine, platelet count, and liver function tests. A validated electronic diagnostic algorithm (Milic et al., Mayo Clin. Proc. 93:1707-19 (2018)) was used to determine the presence of any HDP and type based on accepted clinical criteria and the diagnosis of hypertension, which to required blood pressure elevations in greater than 50% of blood pressure readings (the “50% rule”). The algorithm was validated by comparison of algorithm-based diagnoses to the gold standard, i.e., physician-made diagnoses. The algorithm-based approach demonstrated significant improvements in sensitivity and specificity in the classification of exposure (i.e., HDP) compared to methods that utilized diagnostic codes only. The definition of each HDP type used in the algorithm is described in Table 2. In addition to the data that were required to classify pregnancies as normotensive vs. HDP, demographic, prenatal and intrapartum data were also collected. All abstracted data were first recorded on paper forms and subsequently entered into a database for future analysis.
There were six abstractors involved in retrieving data from the medical records over the course of the project. Both inter-rater and intra-rater reliability were assessed for the diagnosis of HDP and type. For example, there were 21 pregnancies abstracted by the same nurse on different days, several months apart. The diagnosis of HDP and type were the same for all 21 pregnancies, resulting in 100% intra-rater agreement. Additionally, 128 pregnancies were accessed by two different abstractors, of which 126 (98.4%) had the same diagnosis of HDP and type.
For each woman with a pregnancy complicated by HDP, the index date was defined as the date when she first met criteria for HPD. Two referent women with normotensive pregnancies were randomly identified from the women in the birth cohort matched for the date of delivery (±1 year), maternal age (±1 year), and parity at index pregnancy (1 or >1) who had not met criteria for HPD before the index date. Women who first met criteria for HPD based on a delivery prior to 1976 were excluded from this analysis. Women with more than one normotensive pregnancy during 1976-1982 could have been identified as a matched referent for more than one HDP woman.
Chronic conditions recommended by the US Department of Health and Human Services (DHHS) were considered for long-term multimorbidity (Table 3). The following 4 DHHS conditions were excluded because they were rare in the population: human immunodeficiency virus infections, autism spectrum disorders, schizophrenia, and hepatitis. These sixteen conditions were ascertained electronically by retrieving diagnosis codes from inpatient and outpatient visits to REP-affiliated providers from the index date through the women's last visits. To reduce the risk of false-positive diagnoses, a woman was classified as having a condition if she had at least 2 occurrences of a diagnosis code (either the same code or 2 different codes within the code set for a given condition) separated by more than 30 days, as described elsewhere (Rocca et al., Mayo Clin. Proc. 91:1577-89 (2016)). In addition to examining the relationship between HDP and each US DHHS condition, risk of multimorbidity (a measure of accelerated aging) and death were also examined. To capture those conditions that caused acute deaths, a single diagnosis code listed anywhere on a death certificate was also sufficient.
No maternal deaths, strokes, new diagnoses of CAD, CHF or arrhythmias, or initiation of dialysis were reported. Six women with HDP were admitted to the ICU with days in ICU ranging from 2-13; one referent women was admitted to the ICU for 27 days. Four-women (all for the HDP group), developed acute kidney injury, manifested as either oliguria (i.e., urinary output of <500 mL/24 hours) or as an increase in serum creatinine by ≥0.3 mg/dL. Renal function normalized in all cases, and no woman required renal replacement therapy. Finally, women with HDP, compared to referent women, were more likely to receive a blood transfusion in the postpartum period: 38 of 57 (6.7%) versus 21 of 114 (1.8%0, respectively (p-value of <0.001).
Per-pregnancy HPD incidence (per 100 pregnancies) was calculated considering each pregnancy as a distinct event. Per-pregnancy incidence rates were calculated overall, by HPD subtype, and stratified by calendar year and age (<20, 20-24, 25-29, 30-34, and ≥35 years) within each subtype of HPD. The denominators used to determine the incidence, by age and calendar year, are shown in Table 4. For the per-pregnancy incidence rates, 95% confidence intervals (CI) were constructed using a Wilson score interval appropriate for a proportion estimated from clustered binary data given that women could have multiple pregnancies in the cohort. The 95% CIs for the per-woman incidence rates were constructed using an exact method for a binomial proportion. Per-woman HPD incidence (per 100 women) was calculated based on classifying each woman using the following hierarchy: Eclampsia>Preeclampsia superimposed on chronic HTN>Preeclampsia>Chronic HTN>Gestational HTN>Normotensive.
Each of the 16 chronic conditions was evaluated separately, and women with the condition prior to the index date (i.e., for each matched set, the date of the exposed woman's first pregnancy complicated by HPD) were excluded from each analysis in order to evaluate de novo conditions. The duration of follow-up was calculated from the index date to the date of the condition diagnosis, last visit to a REP-affiliated provider prior to the end of the study (Dec. 31, 2017), or subsequent HPD diagnosis for the referent women. Cumulative incidence curves were estimated using the Kaplan-Meier method. Cox proportional hazards models were used to estimate hazard ratios (HRs) and corresponding 95% CIs using age as the time scale with women entering the risk set at their respective index ages. The accumulation of chronic conditions was calculated as the mean number of conditions accumulated over the follow-up after the index date and was represented graphically using Aalen-Johansen curves. Hazard ratios were computed using Anderson-Gill regression models with age as the time scale. Robust sandwich covariance estimates were used to account for either women included in both cohorts (e.g. referent women with subsequent HPD) or women with multiple pregnancies who were selected as referents more than once. Both unadjusted models and models adjusted for education, smoking, and obesity were fit. Tests of statistical significance were conducted at the two-tailed alpha level of 0.05. Statistical analyses were performed using the SAS version 9.4 software package (SAS Institute, Inc.; Cary, NC) and R software v3.4.2.
7,544 mothers were identified who had 9,862 pregnancies of ≥20 weeks' gestation between Jan. 1, 1976 and Dec. 31, 1982 while residents of Olmsted County, Minnesota for the calculation of incidence per-pregnancy (
The age-specific incidence (per 100 pregnancies) of each HDP subtype is shown in
Demographics and perinatal characteristics of HDP pregnancies are shown in Table 7. Pregnancies with preeclampsia or preeclampsia superimposed on chronic hypertension—compared to pregnancies with gestational hypertension—were less frequently carried to term, resulted more frequently in small-for-gestational age infants, and had higher frequencies of stillbirths.
The development of chronic conditions after a pregnancy was studied in 571 women with pregnancies complicated by HDP, and 1142 age- and parity-matched referents (Table 8). The median age at last follow up was not different between the groups: 60.38 years (IQR, 52.64-64.96) for women with HDP and 59.26 (IQR, 36.79-64-47) for referent women. The median length of follow-up was 36.2 years (IQR, 23.5-38.2) and 35.8 years (IQR, 13.7-37.9) for women with a history of HDP and referent women, respectively. Women with a history of HDP, compared to referent women, demonstrated increased risks of cardiac arrhythmias (HR 1.62; 95% CI 1.28-2.05), CAD (HR 1.89; 95% CI 1.26-2.82), stroke (HR 2.27; 95% CI 1.37-3.76), hyperlipidemia (HR 1.27; 95% CI 1.08-1.49), hypertension (HR 2.50; 95% CI 2.08-3.01), diabetes (HR 1.55; 95% CI 1.26-1.91), and CKD (HR 2.41; 95% CI 1.54-3.78) in analyses adjusted for education, smoking, and obesity (Table 8 and
Together these results demonstrate that women with a history of HDP, compared to those with normotensive pregnancies, demonstrated significant increased risks for both kidney and heart disease, including cardiac arrhythmias, CAD, and stroke even at a relatively young median age.
Women with preeclampsia and normotensive pregnant women were recruited from the Mayo Clinic Family Birth Center. All women delivered by clinically indicated Cesarean section. This study was approved by the Mayo Clinic Institutional Review Board and all participants provided written informed consent prior to participating. Each participant's medical record was reviewed to confirm the pregnancy diagnosis and obtain information on demographic characteristics and pregnancy outcomes. The diagnosis of preeclampsia was based on the presence of the following criteria: hypertension after 20 weeks gestation, defined as a blood pressure≥140/90 mmHg; b) proteinuria, defined as ≥300 mg of protein in a 24-hour urine specimen, and/or protein/creatinine (Cr) ratio of 0.3, and/or 1 +(30 mg/L) dipstick urinalysis in the absence of urinary tract infection. In the absence of proteinuria, the diagnosis of preeclampsia was confirmed if i) any of the following laboratory abnormalities were present: thrombocytopenia<100,000/μL serum Cr>1.1 mg/dL or its doubling, and elevated liver function tests, AST and ALT (>2× ULN); or ii) in the presence of pulmonary edema or cerebral or visual symptoms. Women were classified as having a normotensive pregnancy if they showed no signs of hypertension throughout gestation.
MSC isolation from adipose tissue: Abdominal fat tissue (3-5 g) obtained during C-section was cultured at 37° C./5% CO2 in Advanced MEM media supplemented with 5% platelet lysate (PLTmax, Mill Creek Life Sciences, Rochester, MN), which provides a robust growth medium. The third passage of cells was used for phenotype/function analysis. MSC were positive to CD90, CD44, and CD105, negative to CD34, CD31, and CD45 by Flow Cytometry (FlowSight™, Amnis, Seattle, WA), and were able to transdifferentiate into adipocytes, chondrocytes, and osteocytes. For studies in non-pregnant subjects, MSC were isolated from three healthy kidney donors at time of kidney donation.
In vitro effects of TNF-alpha on MSC: MSC isolated from healthy kidney donors were treated with vehicle or 20 ng/mL TNF alpha for 24 hours. After incubation, MSC were washed and RNA isolated, and gene expression of inflammatory cytokines was measured using RT-PCR.
MSC function was assessed by proliferative and migrating capabilities. In brief, MSC migratory function was tested using a QCM™ Chemotaxis Cell Migration kit (ECM508, EMD Millipore) and proliferative activity by MTS (Promega). Proliferation and migration were measured at 490 and 560 nm, respectively, using the SynergyMX spectrophotometer (BioTek Instruments, Inc., Winooski, VT), and expressed in optical density (OD) units.
Cell viability was measured using Flow Cytometry for Annexin V.
5 μm-thick sections of frozen subcutaneous adipose tissue were processed following standard protocols. Inflammation was assessed by staining for TNF-alpha (1:100, Santa Cruz Biotechnology) and monocyte chemoattractant protein (MCP)-1 (1:100, Abcam); oxidative stress was evaluated by the in situ production of superoxide anion, and detected by fluorescence microscopy using dihydroethidium (DHE). Image analysis utilized a computer-aided image-analysis program (AxioVision Carl Zeiss Micro Imaging, Thornwood, NY). Results were expressed as percent of the field-of-view staining (average of 4-6 fields).
Apoptosis was assessed by Annexin V reagent (Essen Bioscience) using the IncuCyte S3 Live-Cell Analysis System (Essen Bioscience).
Angiogenic potential of MSC was assessed using human umbilical vein endothelial cells (HUVEC) angiogenesis assay. NP-MSC and PE-MSC were transferred to a 96-well plate (Corning Incorporated, USA) at 4,000 cells per well where they were co-cultured with previously seeded GFP-expressing HUVEC (IncuCyte CytoLight Green HUVEC Cells) and human fibroblasts (IncuCyte NHDF Cells) as instructed in the manufacturer's kit. The plate was placed in the IncuCyte S3 Live-Cell Analysis System where real-time images were captured every 3 hours. Angiogenesis was assessed as the total network length (mm/mm2) using IncuCyte S3 Software (Essen Bioscience) and compared between groups.
For SABG staining, 50,000 MSC were seeded in a 12-well plate and left until they had reached 70-80% confluency. Cells were fixed in β-gal fixation solution for 10 minutes and washed twice with PBS. Subsequently, the cells were stained overnight using SABG reagent (Cell Signaling Technology) according to the manufacturer's instruction. Nuclei for DAPI imaging were stained using Hoechst reagent. Image analysis utilized a computer-aided image-analysis program (AxioVision Carl Zeiss Micro Imaging, Thornwood, NY). Results are shown presented as percent of the stained cells in field-of-view (average 8-10 fields).
MSC were collected and stored at −80° C. until further use. RNA was isolated by QIAzol Lysis Reagent and RNeasy Mini Columns (QIAGEN, Valencia, CA) following the manufacturer's instructions. RNA concentration and 230/260 absorbance ratios were checked using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). cDNA was synthesized and qPCR was performed using TaqMan™ Fast Advanced Master Mix on Biorad CXF96 Platform in a 10 μL volume using the following thermal protocol: 50° C. for 2 minutes, and 45 cycles of 95° C. for 20 seconds, and 60° C. for 30 seconds. Gene expression was normalized to TATA-box-binding protein (TbP). The following primers were purchased from Applied Biosciences: total p16 (catalog number: Hs00923894), p21 (catalog number: Hs00355782), IL-6 (catalog number: Hs00174131), IL-8 (catalog number: Hs00174103), MCP-1 (catalog number: Hs00234140), PAI-1 (catalog number: Hs01126607), and PAI-2 (catalog number: Hs00299953).
The initial dose response experiments were performed to determine the optimal Dasatinib concentration using the apoptotic assay and IncuCyte S3 Live-Cell Analysis System (Essen Bioscience). Approximately 1×106 MSC (PE and NP) in passage #4, were treated with the senolytic drug, Dasatinib at concentrations of 1, 2, 5 and 10 μM (dissolved in 0.1% DMSO) for 24 hours. Three groups were analyzed: 1) cells incubated in media, 2) vehicle: cells treated with 0.1% DMSO, 3) cells treated with Dasatinib. The MSC were seeded at 5,000 cells/well in 96-well plates (Advanced MEM with 10% FBS) and treated with Dasatinib at increasing concentrations. Annexin V, added at the beginning of the treatment, labeled apoptotic cells yielding red fluorescence. The plate was scanned at a magnification of 10× and degree of fluorescence and images were assessed and taken in real time from the beginning of the treatment up to 24 hours post treatment. Using IncuCyte S3 Software a Red Object Count was generated per well at each time point. Ratios of apoptotic cells in the Dasatinib-treated, vehicle-treated, and media groups were compared and used to determine that the optimal concentration of Dasatinib is 1 μM (see Results) for studying effects of this senolytic agent on the burden of senescent PE-MSC (SABG, SASP, p16, and p21) and their functional angiogenic potential.
Descriptive statistics of demographic and clinical characteristics are reported as mean±SD, median and interquartile range (IQR), or number and percentage, as appropriate. Group differences between women with normotensive pregnancy and those with preeclampsia were determined by the Student t-test or and ANOVA for repeated measurements. Correlations were analyzed using Pearson correlation coefficient. Graphics for plotting individual level data were created using an interactive graph tool and GraphPad Prism 8 (RRID:SCR_002798). Correlations among various parameters were analyzed using Pearson correlation or the Spearman correlation coefficient. All data analyses were performed using SPSS statistical software, version 25 (IBM SPSS, Chicago, IL, RRID:SCR_002865), with significance determined on the basis of α=0.05.
Maternal age did not differ between women with preeclampsia and those with normotensive pregnancy. Women with preeclampsia delivered earlier in pregnancy compared to women with normotensive pregnancies, and, as expected, had higher systolic and diastolic blood pressures (Table 9). Gestational diabetes and twin pregnancy, known risk factors for preeclampsia, were each documented in 20% of the PE pregnancies. Six of 10 preeclamptic pregnancies had clinical evidence of co-existing HELLP (hemolysis, elevated liver enzymes, low platelet count) syndrome
Abdominal fat tissue was obtained from three healthy kidney donors aged 39±3.3 years and a body mass index of 26.6±0.9 (mean±SEM) at time of kidney donation. MSC were isolated and characterized as described in Methods. After co-incubation with vehicle or TNF-alpha (20 ng/mL) for 24 hours, expression of the inflammatory cytokines Interleukin (IL)-6, IL-8, and MCP-1, were significantly elevated in the TNF-alpha compared to the vehicle-treated MSC (
Adipose tissue staining revealed higher expression of TNF-alpha and MCP-1 in preeclampsia compared to normotensive pregnancy (Table 10,
Cell viability was reduced in preeclampsia (Table 10). Women with preeclampsia had a lower percentage of live MSC cells (p=0.012) and a higher percentage of dead cells (p=0.019) than normotensive pregnant women (
Isolated PE-MSC and NP-MSC, from preeclamptic and normotensive pregnant patients respectively, were stained for SABG. The number of senescent cells, as determined by SABG staining, was significantly higher in PE-MSC, with approximately 60.8±14.3% of counted cells being senescent, compared to 2.8±1.3% of the NP-MSC (p<0.001) (
In order to examine and compare the angiogenic potential of MSC, the total network length (mm/mm2) of endothelial cells developed during co-culture with MSC was measured. PE-MSC exhibited lower angiogenic potential compared to their normotensive counterparts (p<0.001) when incubated in the medium (
To determine the optimal senolytic drug concentration, both PE and NP-MSC were treated with four different concentrations of Dasatinib: 1 μM, 2 μM, 5μM and 10 μM. Accumulation of apoptotic bodies (Red Object Count) was assayed after 24 hours treatment in Incucyte. PE-MSC were prone to apoptosis when treated with lower concentrations of Dasatinib. Increasing concentrations of the drug did not result in a further increase in apoptosis. At the same time, NP-MSC were more sensitive to the apoptotic effects of the drug when treated with higher concentrations of Dasatinib (
To test whether treatment with Dasatinib improves the angiogenic potential of PE-MSC, the cells were treated with this senolytic drug as described above. After the treatment, cells were co-cultured with green labelled HUVEC and total network length development was monitored for a total of 8 days (
To demonstrate that Dasatinib can remove cells with the senescence phenotype, both PE-MSC and NP-MSC were stained for SABG before and after treatment with Dasatinib. The treatment with Dasatinib treatment completely removed SABG-stained cells from the culture of PE-MSC (non-treated PE-MSC=62.5±19.5% vs. treated PE-MSC=18.7±8.1%, p<0.0001) (mean±SD) (
Together these results demonstrate that a pro-inflammatory milieu of the abdominal tissue—where MSCs reside—is associated with MSC senescence, and a decrease in MSC-mediated angiogenic effects, and an increase in SASP components. These results also demonstrate that senolytics can be used to improve angiogenesis in MSCs and to decrease expression of SASP components in MSCs.
13 NP and 11 PE historic serum samples collected at the time of delivery were used. SASP components were tested using the Luminex 200 system (Luminex by Eve Technologies Corp.). Women with PE pregnancies demonstrated higher levels of MCP-1, TNF-α, and plasminogen activator inhibitor (PAI)-1 (expressed in ng/mL) compared to women with NP (mean±SD: 316.95±163.95 vs. 207.53±84.78, P=.04; 1.32±0.82 vs. 0.77±0.19, P=0.03; and 58,034.73±18,854.22 vs. 38,117.15±20,856.52; P=0.02, respectively).
These results demonstrate that upregulation of SASP components continues after preeclamptic pregnancies, and can contribute to age-related comorbidities of preeclampsia such as systemic inflammation, and vascular injury and disease.
The gene expression level of senescence markers in the serum of normotensive and preeclamptic patients at delivery was determined as described in Example 3.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Patent Application Ser. No. 62/887,491, filed on Aug. 15, 2019. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
This invention was made with government support under AG13925 and HL136348 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/046299 | 8/14/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62887491 | Aug 2019 | US |
