Kirkland, James L

Key Points This study reveals the temporal development and progression of senescence in an arteriovenous fistula of uremic mice. Senescent genes including p21 increased 7 days after arteriovenous fistula placement, and p16 and phosphorylated p53 cells accumulate between days 14 and 28. Transcriptomic analyses confirm established genes in arteriovenous fistula failure and suggest a collection of novel genes of interest. Background Hemodialysis therapy for ESKD requires suitable vascular access, most commonly the arteriovenous fistula (AVF). Forty percent of AVFs fail within 1 year, leading to morbidity and care costs. Failure mechanisms remain unclear, and current therapies are inadequate. Cellular senescence may be critical in AVF failure, and ESKD itself may accelerate senescence and subsequent vascular dysfunction. Senescence follows AVF placement in rodents with normal kidney function, but the temporal and spatial features in uremic conditions are unknown. This study aimed to characterize AVF changes in uremic mice over time and versus control vessels. Methods Six- to eight-week-old male C57BL6/J mice underwent five/six nephrectomy. Twenty-eight days later, an AVF was created by cuff anastomosis (right carotid artery to the right jugular vein). Transcriptomic analysis was performed 7 days post-AVF, and further samples were collected at 14 and 28 days post-AVF creation for histologic assessment. Results AVF outflow vein morphometry showed reduced neointimal cell density. Whole transcriptomic analysis of outflow versus control veins at 7 days post-AVF placement revealed 1187 upregulated and 3256 downregulated genes. Differentially expressed genes were significantly enriched in two established senescence-related gene sets, SenMayo (a curated panel of approximately 125 genes validated across species to capture senescence and associated proteins) and SenSig (a broader panel assessed to represent fibrotic and stress-induced cell response). Histologically, senescence markers p16, p21, and phospho53 increased between days 14 and 28. Genes common to our dataset, SenMayo, and SenSig were validated with quantitative reverse transcription PCR. Conclusions These data support established markers of AVF failure, such as matrix-metalloproteinases and monocyte chemokines, and identify potential novel modulators of AVF survival that may inform senolytic strategies to improve patency. In summary, this study reveals for the first time the chronologic progression of vascular senescence in the AVF of uremic mice.

Key Points Activin A is implicated in profibrotic and prosenescent kidney injury and correlates with kidney injury markers in animals and humans. Follistatin, through activin A antagonism, reduces senescence burden, macrophage infiltration, and proinflammatory pathway activation in murine diabetic kidney disease. Follistatin and other antagonists of activin A signaling pathways may be promising, novel therapeutics for diabetic kidney disease. Background Circulating activin A, an inflammatory mediator implicated in profibrotic kidney injury and cellular senescence-induced adipose tissue dysfunction, is increased in human diabetic kidney disease (DKD) and directly correlates with kidney dysfunction. We tested the hypothesis that activin A increases kidney injury, senescent cell abundance, and macrophage infiltration in DKD and antagonism through follistatin (FS) therapy diminishes these effects. Methods An accelerated nephropathy type 2 diabetes (db/db) mouse model was generated by implantation of angiotensin II-loaded osmotic minipumps resulting in increased albuminuria and glomerular and tubular injury. Kidney repair effects of FS (5 µ g intraperitoneal; two doses) were assessed through markers of kidney injury, fibrosis, inflammation, cellular senescence, and macrophage infiltration. In vitro studies examined antiactivin effects of FS on high glucose-exposed human monocytes, renal fibroblasts, and renal tubule epithelial cells. Results Activin A antagonism with FS reduced senescence (p19), proinflammatory (including senescence-associated secretory phenotype), and profibrotic markers including activin A. FS improved kidney morphology, restored podocyte markers (nephrin and Wilms tumor-1), and reduced kidney injury biomarkers, albuminuria and kidney fibrosis. FS decreased kidney macrophage and leukocyte infiltration and absent in melanoma 2 inflammasome activation. FS seemed to suppress inflammation through the toll-like receptor-4/NF kappa-light-chain-enhancer of activated B cells pathway in vivo further supported in human macrophages in vitro . In addition, FS reduced hyperglycemia-induced renal fibroblast activation and renal tubule epithelial cell senescence in vitro . Conclusions Activin A is a mediator of kidney injury through macrophage-associated inflammation in murine DKD. FS acts through senomorphic activities which inhibit profibrotic, proinflammatory, and prosenescence signaling by activin A. Hence, antiactivin targeting may aid in the development of a promising, novel therapeutic for DKD.

Modulating immune function is a critical strategy in cancer and atherosclerosis treatments. For cancer, boosting or maintaining the immune system is crucial to prevent tumor growth. However, in vascular disease, mitigating immune responses can decrease inflammation and slow atherosclerosis progression. Anti-inflammatory therapy, therefore, presents a unique dilemma for cancer survivors: while it may decrease cardiovascular risk, it might also promote cancer growth and metastasis by suppressing the immune response. Senescence presents a potentially targetable solution to this challenge; senescence increases the risk of both cancer therapy resistance and vascular disease. Exercise, notably, shows promise in delaying this premature senescence, potentially improving cancer outcomes and lowering vascular disease risk post-treatment. This review focuses on the long-term impact of cancer therapies on vascular health. We underscore the importance of modulating senescence to balance cancer treatment’s effectiveness and its vascular impact, and we emphasize investigating the role of exercise-mediated suppression of senescence in improving cancer survivorship.

Key Points The rat arteriovenous fistula (AVF) model exhibits marked upregulation of p16 Ink4a and p21 Cip1 and multiple markers of senescence. Fisetin, an established vasoprotective senolytic agent, when administered for 3 weeks, increases AVF blood flow and AVF outward remodeling. Heme is shown to be a novel prosenescence metabolite, and when chronically administered, it decreases AVF blood flow. Background Maturational failure of dialysis arteriovenous fistulas (AVFs) not uncommonly occurs and is of considerable and timely importance. Our prior studies demonstrate that senescence, a phenotypic process that promotes vascular and other diseases, occurs in the murine AVF. In this study, we examined whether senescence also occurs in the rat AVF model and the effect of compounds that inhibit or accelerate senescence. Methods The rat AVF was created in the femoral vessels by an end vein-side artery anastomosis. In the AVF, we assessed the expression of critical drivers of senescence, specifically, the cell cycle inhibitors p16 Ink4a and p21 Cip1 , and such indices of a senescence phenotype as senescence-associated β -galactosidase (SA- β -gal) activity, SA- β -gal staining, and a senescence-associated secretory phenotype. We examined the effects of compounds that retard or accelerate senescence on AVF blood flow. Results The AVF evinced upregulation of p16 Ink4a and p21 Cip1 when assessed 3 days after AVF creation. The AVF also demonstrated increased SA- β -gal activity in the artery and vein; staining for SA- β -gal in the AVF artery, anastomosis, and vein; and a prominent senescence-associated secretory phenotype. Fisetin, an established senolytic that is protective in other models of vascular injury, when administered for 3 weeks, increased AVF blood flow and outward remodeling. Hemin, when administered for 3 weeks, decreased AVF blood flow. We demonstrate that hemin is a novel inducer of a senescence phenotype in endothelial cells, as reflected by several senescence indices. However, when administered relatively acutely (for 5 days), hemin increased AVF blood flow by heme oxygenase–dependent mechanisms because the latter was entirely prevented by a competitive inhibitor of heme oxygenase activity. Conclusions The rat AVF exhibits senescence within 3 days of its creation. Chronic administration of a senolytic compound (fisetin) increases AVF blood flow, whereas chronic administration of a prosenescence compound (hemin) decreases AVF blood flow.

Background Coronary microvascular dysfunction (CMD) represents an early functional characteristic of coronary vascular aging. Klotho (α‐klotho) is a circulating protein inversely linked to physiological aging. We examined low klotho as a potential marker for vascular aging in patients with CMD and no coronary artery disease. Methods and Results Patients undergoing nonurgent angiogram for chest pain who had no coronary artery disease underwent invasive coronary microvascular and endothelial function testing. CMD was defined by ≤50% increase in coronary blood flow (percentage change in coronary blood flow) in response to intracoronary acetylcholine or coronary flow reserve ≤2. Fresh arterial whole blood was used to analyze circulating endothelial progenitor cells with flow cytometry. Stored arterial plasma was used for klotho analysis by ELISA. Participants with CMD (n=62) were compared with those without CMD (n=36). Those with CMD were age 55±10 years (versus 51±11 years; P =0.07) and 73% women (versus 81%; P =0.38). Traditional risk factors for coronary artery disease were similar between groups. Patients with CMD had less klotho (0.88±1.50 versus 1.75±2.38 ng/mL; P =0.03), and the odds of low klotho in CMD were significant in a logistic regression model after adjusting for traditional cardiovascular risk factors (odds ratio [OR], 0.80 [95% CI, 0.636–0.996]; P =0.05). Higher klotho was associated with higher numbers of endothelial progenitor cells with vascular regenerative potential (CD34 + and CD34 + CD133 + KDR + ). Among a subgroup of patients with atherosclerotic cardiovascular disease risk <5% (n=58), CMD remained associated with lower klotho (OR, 0.80 [95% CI, 0.636–0.996]; P =0.047). Conclusions Klotho may be a biomarker for CMD and may be a therapeutic target for groups of patients without significant traditional cardiovascular risk.

Peripartum cardiomyopathy (PPCM) is an idiopathic form of pregnancy-induced heart failure associated with preeclampsia. Circulating factors in late pregnancy are thought to contribute to both diseases, suggesting a common underlying pathophysiological process. However, what drives this process remains unclear. Using serum proteomics, we identified the senescence-associated secretory phenotype (SASP), a marker of cellular senescence associated with biological aging, as the most highly up-regulated pathway in young women with PPCM or preeclampsia. Placentas from women with preeclampsia displayed multiple markers of amplified senescence and tissue aging, as well as overall increased gene expression of 28 circulating proteins that contributed to SASP pathway enrichment in serum samples from patients with preeclampsia or PPCM. The most highly expressed placental SASP factor, activin A, was associated with cardiac dysfunction or heart failure severity in women with preeclampsia or PPCM. In a murine model of PPCM induced by cardiomyocyte-specific deletion of the gene encoding peroxisome proliferator–activated receptor γ coactivator-1α, inhibiting activin A signaling in the early postpartum period with a monoclonal antibody to the activin type II receptor improved heart function. In addition, attenuating placental senescence with the senolytic compound fisetin in late pregnancy improved cardiac function in these animals. These findings link senescence biology to cardiac dysfunction in pregnancy and help to elucidate the pathogenesis underlying cardiovascular diseases of pregnancy.

Abstract Multiple changes occur in hormonal regulation with aging and across various endocrine organs. These changes are associated with multiple age-related disorders and diseases. A better understanding of responsible underling biological mechanisms could help in the management of multiple endocrine disorders over and above hormone replacement therapy (HRT). Cellular senescence is involved in multiple biological aging processes and pathologies common in elderly individuals. Cellular senescence, which occurs in many older individuals but also across the lifespan in association with tissue damage, acute and chronic diseases, certain drugs, and genetic syndromes, may contribute to such endocrine disorders as osteoporosis, metabolic syndrome, and type 2 diabetes mellitus. Drugs that selectively induce senescent cell removal, “senolytics,”, and drugs that attenuate the tissue-destructive secretory state of certain senescent cells, “senomorphics,” appear to delay the onset of or alleviate multiple diseases, including but not limited to endocrine disorders such as diabetes, complications of obesity, age-related osteoporosis, and cancers as well as atherosclerosis, chronic kidney disease, neurodegenerative disorders, and many others. More than 30 clinical trials of senolytic and senomorphic agents have already been completed, are underway, or are planned for a variety of indications. Targeting senescent cells is a novel strategy that is distinct from conventional therapies such as HRT, and thus might address unmet medical needs and can potentially amplify effects of established endocrine drug regimens, perhaps allowing for dose decreases and reducing side effects.

Abstract Senescent cell accumulation contributes to the progression of age-related disorders including Alzheimer’s disease (AD). Clinical trials evaluating senolytics, drugs that clear senescent cells, are underway, but lack standardized outcome measures. Our team recently published data from the first open-label trial to evaluate senolytics (dasatinib plus quercetin) in AD. After 12-weeks of intermittent treatment, we reported brain exposure to dasatinib, favorable safety and tolerability, and modest post-treatment changes in cerebrospinal fluid (CSF) inflammatory and AD biomarkers using commercially available assays. Herein, we present more comprehensive exploratory analyses of senolytic associated changes in AD relevant proteins, metabolites, lipids, and transcripts measured across blood, CSF, and urine. These analyses included mass spectrometry for precise quantification of amyloid beta (Aß) and tau in CSF; immunoassays to assess senescence associated secretory factors in plasma, CSF, and urine; mass spectrometry analysis of urinary metabolites and lipids in blood and CSF; and transcriptomic analyses relevant to chronic stress measured in peripheral blood cells. Levels of Aß and tau species remained stable. Targeted cytokine and chemokine analyses revealed treatment-associated increases in inflammatory plasma fractalkine and MMP-7 and CSF IL-6. Urinary metabolites remained unchanged. Modest treatment-associated lipid profile changes suggestive of decreased inflammation were observed both peripherally and centrally. Blood transcriptomic analysis indicated downregulation of inflammatory genes including FOS, FOSB, IL1β, IL8, JUN, JUNB, PTGS2. These data provide a foundation for developing standardized outcome measures across senolytic studies and indicate distinct biofluid-specific signatures that will require validation in future studies. ClinicalTrials.gov: NCT04063124.

Key Points In heme protein–mediated AKI (HP-AKI), a senescence phenotype promptly occurs, and increased expression of p16 Ink4a contributes to HP-AKI. Renal p16 Ink4a expression is induced by hemoglobin, myoglobin, and heme in vivo and in renal epithelial cells exposed to heme in vitro . Impairing the binding or degradation of heme by hemopexin deficiency or heme oxygenase-1 deficiency, respectively, further upregulates p16 Ink4a . Background Understanding the pathogenetic basis for AKI involves the study of ischemic and nephrotoxic models of AKI, the latter including heme protein–mediated AKI (HP-AKI). Recently, interest has grown regarding the role of senescence as a mechanism of kidney injury, including AKI. We examined whether senescence occurs in HP-AKI and potential inducers of and the role of a key driver of senescence, namely, p16 Ink4a , in HP-AKI. Methods The long-established murine glycerol model of HP-AKI was used, and indices of senescence were examined. To evaluate the interaction of heme and p16 Ink4a expression, murine models of genetic deficiency of hemopexin ( HPX ) and heme oxygenase-1 ( HO-1 ) were used. To determine the involvement of p16 Ink4a in HP-AKI, the population of p16 Ink4a -expressing cells was reduced using the INK-ATTAC model. Results Using multiple indices, a senescence phenotype appears in the kidney within hours after the induction of HP-AKI. This phenotype includes significant upregulation of p16 Ink4a . p16 Ink4a is upregulated in the kidney after the individual administration of myoglobin, hemoglobin, and heme, as well as in renal epithelial cells exposed to heme in vitro . Genetic deficiencies of HPX and HO-1 , which, independently, are expected to increase heme content in the kidney, exaggerate induction of p16 Ink4a in the kidney and exacerbate HP-AKI, the latter shown in the present studies involving HPX −/− mice and in previous studies involving HO-1 −/− mice. Finally, reduction in the population of p16 Ink4a -expressing cells in the kidney improves renal function in HP-AKI even within 24 hours. Conclusions The pathogenesis of HP-AKI involves senescence and the induction of p16 Ink4a , the latter driven, in part, by hemoglobin, myoglobin, and heme.

Abstract Cellular senescence has been identified as a pathological mechanism linked to tau and amyloid beta (Aβ) accumulation in mouse models of Alzheimer's disease (AD). Clearance of senescent cells using the senolytic compounds dasatinib (D) and quercetin (Q) reduced neuropathological burden and improved clinically relevant outcomes in the mice. Herein, we conducted a vanguard open-label clinical trial of senolytic therapy for AD with the primary aim of evaluating central nervous system (CNS) penetrance, as well as exploratory data collection relevant to safety, feasibility, and efficacy. Participants with early-stage symptomatic AD were enrolled in an open-label, 12-week pilot study of intermittent orally-delivered D+Q. CNS penetrance was assessed by evaluating drug levels in cerebrospinal fluid (CSF) using high performance liquid chromatography with tandem mass spectrometry. Safety was continuously monitored with adverse event reporting, vitals, and laboratory work. Cognition, neuroimaging, and plasma and CSF biomarkers were assessed at baseline and post-treatment. Five participants (mean age: 76±5 years; 40% female) completed the trial. The treatment increased D and Q levels in the blood of all participants ranging from 12.7 to 73.5 ng/ml for D and 3.29-26.30 ng/ml for Q. D levels were detected in the CSF of four participants ranging from 0.281 to 0.536 ng/ml (t(4)=3.123, p=0.035); Q was not detected. Treatment was well-tolerated with no early discontinuation and six mild to moderate adverse events occurring across the study. Cognitive and neuroimaging endpoints did not significantly differ from baseline to post-treatment. CNS levels of IL-6 and GFAP increased from baseline to post-treatment (t(4)=3.913, p=008 and t(4)=3.354, p=0.028, respectively) concomitant with decreased levels of several cytokines and chemokines associated with senescence, and a trend toward higher levels of Aβ42 (t(4)=-2.338, p=0.079). Collectively the data indicate the CNS penetrance of D and provide preliminary support for the safety, tolerability, and feasibility of the intervention and suggest that astrocytes and Aβ may be particularly responsive to the treatment. While early results are promising, fully powered, placebo-controlled studies are needed to evaluate the potential of AD modification with the novel approach of targeting cellular senescence.