Targeting Cell Senescence and Senolytics: Novel Interventions for Age-Related Endocrine Dysfunction.

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.

Potential Clinical Implications of Senotherapies for Cardiovascular Disease.

Aging is a major risk factor for cardiovascular diseases (CVDs) and accumulating evidence indicates that biological aging has a significant effect on the onset and progression of CVDs. In recent years, therapies targeting senescent cells (senotherapies), particularly senolytics that selectively eliminate senescent cells, have been developed and show promise for treating geriatric syndromes and age-associated diseases, including CVDs. In 2 pilot studies published in 2019 the senolytic combination, dasatinib plus quercetin, improved physical function in patients with idiopathic pulmonary fibrosis and eliminated senescent cells from adipose tissue in patients with diabetic kidney disease. More than 30 clinical trials using senolytics are currently underway or planned. In preclinical CVD models, senolytics appear to improve heart failure, ischemic heart disease, valvular heart disease, atherosclerosis, aortic aneurysm, vascular dysfunction, dialysis arteriovenous fistula patency, and pre-eclampsia. Because senotherapies are completely different strategies from existing treatment paradigms, they might alleviate diseases for which there are no current effective treatments or they could be used in addition to current therapies to enhance efficacy. Moreover, senotherapies might delay, prevent, alleviate or treat multiple diseases in the elderly and reduce polypharmacy, because senotherapies target fundamental aging mechanisms. We comprehensively summarize the preclinical evidence about senotherapies for CVDs and discuss future prospects for their clinical application.

Endothelial SIRT-1 has a critical role in the maintenance of capillarization in brown adipose tissue.

Brown adipose tissue (BAT) has critical roles in thermogenesis and systemic metabolism. Capillary rarefaction was reported to develop in BAT with dietary obesity, and previous studies showed that suppression of vascular endothelial growth factor A (VEGF-A) reduced capillary density in BAT, promoting the functional decline of this organ. Capillarization is regulated through the balance between angiogenesis and vasculogenesis on the one hand and apoptosis of endothelial cells (ECs) on the other; however, the role of EC apoptosis in BAT remained to be explored. In studies testing the role of boysenberry polyphenols (BoyP) in BAT, we found that BoyP decreased EC apoptosis, enhanced capillarization in BAT, and ameliorated dietary BAT dysfunction, which was associated with the upregulation of nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin 1 (SIRT-1) in ECs. Our studies suggest that EC SIRT-1 would be one of the potential targets of BoyP that contributes to BAT capillarization and function.

Glycoprotein nonmetastatic melanoma protein B regulates lysosomal integrity and lifespan of senescent cells.

Accumulation of senescent cells in various tissues has been reported to have a pathological role in age-associated diseases. Elimination of senescent cells (senolysis) was recently reported to reversibly improve pathological aging phenotypes without increasing rates of cancer. We previously identified glycoprotein nonmetastatic melanoma protein B (GPNMB) as a seno-antigen specifically expressed by senescent human vascular endothelial cells and demonstrated that vaccination against Gpnmb eliminated Gpnmb-positive senescent cells, leading to an improvement of age-associated pathologies in mice. The aim of this study was to elucidate whether GPNMB plays a role in senescent cells. We examined the potential role of GPNMB in senescent cells by testing the effects of GPNMB depletion and overexpression in vitro and in vivo. Depletion of GPNMB from human vascular endothelial cells shortened their replicative lifespan and increased the expression of negative cell cycle regulators. Conversely, GPNMB overexpression protected these cells against stress-induced premature senescence. Depletion of Gpnmb led to impairment of vascular function and enhanced atherogenesis in mice, whereas overexpression attenuated dietary vascular dysfunction and atherogenesis. GPNMB was upregulated by lysosomal stress associated with cellular senescence and was a crucial protective factor in maintaining lysosomal integrity. GPNMB is a seno-antigen that acts as a survival factor in senescent cells, suggesting that targeting seno-antigens such as GPNMB may be a novel strategy for senolytic treatments.

Cardiac mitofusin-1 is reduced in non-responding patients with idiopathic dilated cardiomyopathy.

Prognosis of severe heart failure remains poor. Urgent new therapies are required. Some heart failure patients do not respond to established multidisciplinary treatment and are classified as "non-responders". The outcome is especially poor for non-responders, and underlying mechanisms are largely unknown. Mitofusin-1 (Mfn1), a mitochondrial fusion protein, is significantly reduced in non-responding patients. This study aimed to elucidate the role of Mfn1 in the failing heart. Twenty-two idiopathic dilated cardiomyopathy (IDCM) patients who underwent endomyocardial biopsy of intraventricular septum were included. Of the 22 patients, 8 were non-responders (left ventricular (LV) ejection fraction (LVEF) of < 10% improvement at late phase follow-up). Electron microscopy (EM), quantitative PCR, and immunofluorescence studies were performed to explore the biological processes and molecules involved in failure to respond. Studies in cardiac specific Mfn1 knockout mice (c-Mfn1 KO), and in vitro studies with neonatal rat ventricular myocytes (NRVMs) were also conducted. A significant reduction in mitochondrial size in cardiomyocytes, and Mfn1, was observed in non-responders. A LV pressure overload with thoracic aortic constriction (TAC) c-Mfn1 KO mouse model was generated. Systolic function was reduced in c-Mfn1 KO mice, while mitochondria alteration in TAC c-Mfn1 KO mice increased. In vitro studies in NRVMs indicated negative regulation of Mfn1 by the ß-AR/cAMP/PKA/miR-140-5p pathway resulting in significant reduction in mitochondrial respiration of NRVMs. The level of miR140-5p was increased in cardiac tissues of non-responders. Mfn1 is a biomarker of heart failure in non-responders. Therapies targeting mitochondrial dynamics and homeostasis are next generation therapy for non-responding heart failure patients.

Senolytic vaccination improves normal and pathological age-related phenotypes and increases lifespan in progeroid mice.

Elimination of senescent cells (senolysis) was recently reported to improve normal and pathological changes associated with aging in mice1,2. However, most senolytic agents inhibit antiapoptotic pathways3, raising the possibility of off-target effects in normal tissues. Identification of alternative senolytic approaches is therefore warranted. Here we identify glycoprotein nonmetastatic melanoma protein B (GPNMB) as a molecular target for senolytic therapy. Analysis of transcriptome data from senescent vascular endothelial cells revealed that GPNMB was a molecule with a transmembrane domain that was enriched in senescent cells (seno-antigen). GPNMB expression was upregulated in vascular endothelial cells and/or leukocytes of patients and mice with atherosclerosis. Genetic ablation of Gpnmb-positive cells attenuated senescence in adipose tissue and improved systemic metabolic abnormalities in mice fed a high-fat diet, and reduced atherosclerotic burden in apolipoprotein E knockout mice on a high-fat diet. We then immunized mice against Gpnmb and found a reduction in Gpnmb-positive cells. Senolytic vaccination also improved normal and pathological phenotypes associated with aging, and extended the male lifespan of progeroid mice. Our results suggest that vaccination targeting seno-antigens could be a potential strategy for new senolytic therapies.

p53 plays a crucial role in endothelial dysfunction associated with hyperglycemia and ischemia.

p53 is a guardian of the genome that protects against carcinogenesis. There is accumulating evidence that p53 is activated with aging. Such activation has been reported to contribute to various age-associated pathologies, but its role in vascular dysfunction is largely unknown. The aim of this study was to investigate whether activation of endothelial p53 has a pathological effect in relation to endothelial function. We established endothelial p53 loss-of-function and gain-of-function models by breeding endothelial-cell specific Cre mice with floxed Trp53 or floxed Mdm2/Mdm4 mice, respectively. Then we induced diabetes by injection of streptozotocin. In the diabetic state, endothelial p53 expression was markedly up-regulated and endothelium-dependent vasodilatation was significantly impaired. Impairment of vasodilatation was significantly ameliorated in endothelial p53 knockout (EC-p53 KO) mice, and deletion of endothelial p53 also significantly enhanced the induction of angiogenesis by ischemia. Conversely, activation of endothelial p53 by deleting Mdm2/Mdm4 reduced both endothelium-dependent vasodilatation and ischemia-induced angiogenesis. Introduction of p53 into human endothelial cells up-regulated the expression of phosphatase and tensin homolog (PTEN), thereby reducing phospho-eNOS levels. Consistent with these results, the beneficial impact of endothelial p53 deletion on endothelial function was attenuated in EC-p53 KO mice with an eNOS-deficient background. These results show that endothelial p53 negatively regulates endothelium-dependent vasodilatation and ischemia-induced angiogenesis, suggesting that inhibition of endothelial p53 could be a novel therapeutic target in patients with metabolic disorders.

Role of smooth muscle cell p53 in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by remodeling and narrowing of the pulmonary arteries, which lead to elevation of right ventricular pressure, heart failure, and death. Proliferation of pulmonary artery smooth muscle cells (PASMCs) is thought to be central to the pathogenesis of PAH, although the underlying mechanisms are still being explored. The protein p53 is involved in cell cycle coordination, DNA repair, apoptosis, and cellular senescence, but its role in pulmonary hypertension (PH) is not fully known. We developed a mouse model of hypoxia-induced pulmonary hypertension (PH) and found significant reduction of p53 expression in the lungs. Our in vitro experiments with metabolomic analyses and the Seahorse XF extracellular flux analyzer indicated that suppression of p53 expression in PASMCs led to upregulation of glycolysis and downregulation of mitochondrial respiration, suggesting a proliferative phenotype resembling that of cancer cells. It was previously shown that systemic genetic depletion of p53 in a murine PH model led to more severe lung manifestations. Lack of information about the role of cell-specific p53 signaling promoted us to investigate it in our mouse PH model with the inducible Cre-loxP system. We generated a mouse model with SMC-specific gain or loss of p53 function by crossing Myh11-Cre/ERT2 mice with floxed Mdm4 mice or floxed Trp53 mice. After these animals were exposed to hypoxia for 4 weeks, we conducted hemodynamic and echocardiographic studies. Surprisingly, the severity of PH was similar in both groups of mice and there were no differences between the genotypes. Our findings in these mice indicate that activation or suppression of p53 signaling in SMCs has a minor role in the pathogenesis of PH and suggest that p53 signaling in other cells (endothelial cells, immune cells, or fibroblasts) may be involved in the progression of this condition.

Boysenberry polyphenol inhibits endothelial dysfunction and improves vascular health.

Endothelial cells have an important role in maintaining vascular homeostasis. Age-related disorders (including obesity, diabetes, and hypertension) or aging per se induce endothelial dysfunction that predisposes to the development of atherosclerosis. Polyphenols have been reported to suppress age-related endothelial cell disorders, but their role in vascular function is yet to be determined. We investigated the influence of boysenberry polyphenol on vascular health under metabolic stress in a murine model of dietary obesity. We found that administration of boysenberry polyphenol suppressed production of reactive oxygen species (ROS) and increased production of nitric oxide (NO) in the aorta. It has been reported that p53 induces cellular senescence and has a crucial role in age-related disorders, including heart failure and diabetes. Administration of boysenberry polyphenol significantly reduced the endothelial p53 level in the aorta and ameliorated endothelial cell dysfunction in iliac arteries under metabolic stress. Boysenberry polyphenol also reduced ROS and p53 levels in cultured human umbilical vein endothelial cells (HUVECs), while increasing NO production. Uncoupled endothelial nitric oxide synthase (eNOS monomer) is known to promote ROS production. We found that boysenberry polyphenol reduced eNOS monomer levels both in vivo and in vitro, along with an increase of eNOS dimerization. To investigate the components of boysenberry polyphenol mediating these favorable biological effects, we extracted the anthocyanin fractions. We found that anthocyanins contributed to suppression of ROS and p53, in association with increased NO production and eNOS dimerization. In an ex vivo study, anthocyanins promoted relaxation of iliac arteries from mice with dietary obesity. These findings indicate that boysenberry polyphenol and anthocyanins, a major component of this polyphenol, inhibit endothelial dysfunction and contribute to maintenance of vascular homeostasis.

Catecholamine-Induced Senescence of Endothelial Cells and Bone Marrow Cells Promotes Cardiac Dysfunction in Mice.

Previous studies have suggested that cellular senescence plays a central role in the progression of pathologic changes in the failing heart. It is well known that the sympathetic nervous system is activated in patients with heart failure, and this change is associated with poor clinical outcomes. Sympathetic activation increases the levels of various catecholamines, such as epinephrine and norepinephrine, but the contribution of these catecholamines to cellular senescence associated with heart failure remains to be determined. We found that catecholamine infusion induced senescence of endothelial cells and bone marrow cells, and promoted cardiac dysfunction in mice. In C57BL/6NCr mice, the continuous infusion of isoproterenol-induced cardiac inflammation and cardiac dysfunction. Expression of p53, a master regulator of cellular senescence, was increased in the cardiac tissue and bone marrow cells of these mice. Suppression of cellular senescence by genetic deletion of p53 in endothelial cells or bone marrow cells led to improvement of isoproterenol-induced cardiac dysfunction. In vitro studies showed that adrenergic signaling increased the expression of p53 and adhesion molecules by endothelial cells and macrophages. Our results indicate that catecholamine-induced senescence of endothelial cells and bone marrow cells plays a pivotal role in the progression of heart failure. Suppression of catecholamine-p53 signaling is crucial for inhibition of remodeling in the failing heart.

Amlodipine Inhibits Vascular Cell Senescence and Protects Against Atherogenesis Through the Mechanism Independent of Calcium Channel Blockade.

Vascular cells have a finite lifespan and eventually enter irreversible growth arrest called cellular senescence. We have previously suggested that vascular cell senescence contributes to the pathogenesis of human atherosclerosis. Amlodipine is a mixture of two enantiomers, one of which (S- enantiomer) has L-type channel blocking activity, while the other (R+ enantiomer) shows ~1000-fold weaker channel blocking activity than S- enantiomer and has other unknown effects. It has been reported that amlodipine inhibits the progression of atherosclerosis in humans, but the molecular mechanism of this beneficial effect remains unknown. Apolipoprotein E-deficient mice on a high-fat diet were treated with amlodipine, its R+ enantiomer or vehicle for eight weeks. Compared with vehicle treatment, both amlodipine and the R+ enantiomer significantly reduced the number of senescent vascular cells and inhibited plaque formation to a similar extent. Expression of the pro-inflammatory molecule interleukin-1ß was markedly upregulated in vehicle-treated mice, but was inhibited to a similar extent by treatment with amlodipine or the R+ enantiomer. Likewise, activation of p53 (a critical inducer of senescence) was markedly suppressed by treatment with amlodipine or the R+ enantiomer. These results suggest that amlodipine inhibits vascular cell senescence and protects against atherogenesis at least partly by a mechanism that is independent of calcium channel blockade.

Inhibition of dipeptidyl peptidase-4 ameliorates cardiac ischemia and systolic dysfunction by up-regulating the FGF-2/EGR-1 pathway.

Dipeptidyl peptidase 4 inhibitors are used worldwide in the management of diabetes, but their role in the prevention or treatment of cardiovascular disorders has yet to be defined. We found that linagliptin, a DPP-4 inhibitor, suppressed capillary rarefaction in the hearts of mice with dietary obesity. Metabolomic analysis performed with capillary electrophoresis/mass spectrometry (LC-MS/MS) showed that linagliptin promoted favorable metabolic remodeling in cardiac tissue, which was characterized by high levels of citrulline and creatine. DNA microarray analysis revealed that the cardiac tissue level of early growth response protein 1 (EGR-1), which activates angiogenesis, was significantly reduced in untreated mice with dietary obesity, while this decrease was inhibited by administration of linagliptin. Mature fibroblast growth factor 2 (FGF-2) has a putative truncation site for DPP-4 at the NH2-terminal, and LC-MS/MS showed that recombinant DPP-4 protein cleaved the NH2-terminal dipeptides of mature FGF-2. Incubation of cultured neonatal rat cardiomyocytes with FGF-2 increased Egr1 expression, while it was suppressed by recombinant DPP-4 protein. Furthermore, vascular endothelial growth factor-A had a critical role in mediating FGF-2/EGR-1 signaling. In conclusion, pharmacological inhibition of DPP-4 suppressed capillary rarefaction and contributed to favorable remodeling of cardiac metabolism in mice with dietary obesity.

p53-Induced inflammation exacerbates cardiac dysfunction during pressure overload.

The rates of death and disability caused by severe heart failure are still unacceptably high. There is evidence that the sterile inflammatory response has a critical role in the progression of cardiac remodeling in the failing heart. The p53 signaling pathway has been implicated in heart failure, but the pathological link between p53 and inflammation in the failing heart is largely unknown. Here we demonstrate a critical role of p53-induced inflammation in heart failure. Expression of p53 was increased in cardiac endothelial cells and bone marrow cells in response to pressure overload, leading to up-regulation of intercellular adhesion molecule-1 (ICAM1) expression by endothelial cells and integrin expression by bone marrow cells. Deletion of p53 from endothelial cells or bone marrow cells significantly reduced ICAM1 or integrin expression, respectively, as well as decreasing cardiac inflammation and ameliorating systolic dysfunction during pressure overload. Conversely, overexpression of p53 in bone marrow cells led to an increase of integrin expression and cardiac inflammation that reduced systolic function. Norepinephrine markedly increased p53 expression in endothelial cells and macrophages. Reducing ß2-adrenergic receptor expression in endothelial cells or bone marrow cells attenuated cardiac inflammation and improved systolic dysfunction during pressure overload. These results suggest that activation of the sympathetic nervous system promotes cardiac inflammation by up-regulating ICAM1 and integrin expression via p53 signaling to exacerbate cardiac dysfunction. Inhibition of p53-induced inflammation may be a novel therapeutic strategy for heart failure.

Notch signaling regulates the lifespan of vascular endothelial cells via a p16-dependent pathway.

Evolutionarily conserved Notch signaling controls cell fate determination and differentiation during development, and is also essential for neovascularization in adults. Although recent studies suggest that the Notch pathway is associated with age-related conditions, it remains unclear whether Notch signaling is involved in vascular aging. Here we show that Notch signaling has a crucial role in endothelial cell senescence. Inhibition of Notch signaling in human endothelial cells induced premature senescence via a p16-dependent pathway. Conversely, over-expression of Notch1 or Jagged1 prolonged the replicative lifespan of endothelial cells. Notch1 positively regulated the expression of inhibitor of DNA binding 1 (Id1) and MAP kinase phosphatase 1 (MKP1), while MKP1 further up-regulated Id1 expression by inhibiting p38MAPK-induced protein degradation. Over-expression of Id1 down-regulated p16 expression, thereby inhibiting premature senescence of Notch1-deleted endothelial cells. These findings indicate that Notch1 signaling has a role in the regulation of endothelial cell senescence via a p16-dependent pathway and suggest that activation of Notch1 could be a new therapeutic target for treating age-associated vascular diseases.

Peptides derived from repulsive guidance molecule act as antagonists.

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system [T. Yamashita, B.K. Mueller, K. Hata, Neogenin and RGM signaling in the central nervous system, Curr. Opin. Neurobiol. 17 (2007) 29-34]. Functional studies in Xenopus and chick embryos have revealed the roles of RGM in axon guidance and laminar patterning, while those in mouse embryos have demonstrated its function in regulating the cephalic neural tube closure. Importantly, RGM inhibition enhanced the growth of injured axons and promoted functional recovery after spinal cord injury in rats. Here, we identified two RGMa-derived peptides that functioned as antagonists against RGMa. The peptides studied in vitro dose-dependently suppressed the neurite growth inhibition and growth cone collapse induced by RGMa. Thus, these peptides are promising reagents to treat injuries of the central nervous system.