Myocardial Recovery in Recent Onset Dilated Cardiomyopathy: Role of CDCP1 and Cardiac Fibrosis.

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of heart failure and carries a high mortality rate. Myocardial recovery in DCM-related heart failure patients is highly variable, with some patients having little or no response to standard drug therapy. A genome-wide association study may agnostically identify biomarkers and provide novel insight into the biology of myocardial recovery in DCM. METHODS: A genome-wide association study for change in left ventricular ejection fraction was performed in 686 White subjects with recent-onset DCM who received standard pharmacotherapy. Genome-wide association study signals were subsequently functionally validated and studied in relevant cellular models to understand molecular mechanisms that may have contributed to the change in left ventricular ejection fraction. RESULTS: The genome-wide association study identified a highly suggestive locus that mapped to the 5'-flanking region of the CDCP1 (CUB [complement C1r/C1s, Uegf, and Bmp1] domain containing protein 1) gene (rs6773435; P=7.12×10-7). The variant allele was associated with improved cardiac function and decreased CDCP1 transcription. CDCP1 expression was significantly upregulated in human cardiac fibroblasts (HCFs) in response to the PDGF (platelet-derived growth factor) signaling, and knockdown of CDCP1 significantly repressed HCF proliferation and decreased AKT (protein kinase B) phosphorylation. Transcriptomic profiling after CDCP1 knockdown in HCFs supported the conclusion that CDCP1 regulates HCF proliferation and mitosis. In addition, CDCP1 knockdown in HCFs resulted in significantly decreased expression of soluble ST2 (suppression of tumorigenicity-2), a prognostic biomarker for heart failure and inductor of cardiac fibrosis. CONCLUSIONS: CDCP1 may play an important role in myocardial recovery in recent-onset DCM and mediates its effect primarily by attenuating cardiac fibrosis.

Senolytic Drugs: Reducing Senescent Cell Viability to Extend Health Span.

Senescence is the consequence of a signaling mechanism activated in stressed cells to prevent proliferation of cells with damage. Senescent cells (Sncs) often develop a senescence-associated secretory phenotype to prompt immune clearance, which drives chronic sterile inflammation and plays a causal role in aging and age-related diseases. Sncs accumulate with age and at anatomical sites of disease. Thus, they are regarded as a logical therapeutic target. Senotherapeutics are a new class of drugs that selectively kill Sncs (senolytics) or suppress their disease-causing phenotypes (senomorphics/senostatics). Since 2015, several senolytics went from identification to clinical trial. Preclinical data indicate that senolytics alleviate disease in numerous organs, improve physical function and resilience, and suppress all causes of mortality, even if administered to the aged. Here, we review the evidence that Sncs drive aging and disease, the approaches to identify and optimize senotherapeutics, and the current status of preclinical and clinical testing of senolytics.

Length-independent telomere damage drives post-mitotic cardiomyocyte senescence.

Ageing is the biggest risk factor for cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age-related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post-mitotic cardiomyocytes and investigate whether clearance of senescent cells attenuates age-related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction and crucially can occur independently of cell division and telomere length. Length-independent telomere damage in cardiomyocytes activates the classical senescence-inducing pathways, p21CIP and p16INK4a, and results in a non-canonical senescence-associated secretory phenotype, which is pro-fibrotic and pro-hypertrophic. Pharmacological or genetic clearance of senescent cells in mice alleviates detrimental features of cardiac ageing, including myocardial hypertrophy and fibrosis. Our data describe a mechanism by which senescence can occur and contribute to age-related myocardial dysfunction and in the wider setting to ageing in post-mitotic tissues.

Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan.

Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16(Ink4a) (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16(Ink4a)-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16(Ink4a)-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16(Ink4a)-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

Smooth muscle brain-derived neurotrophic factor contributes to airway hyperreactivity in a mouse model of allergic asthma.

Recent studies have demonstrated an effect of neurotrophins, particularly brain-derived neurotrophic factor (BDNF), on airway contractility [ via increased airway smooth muscle (ASM) intracellular calcium [Ca2+]i] and remodeling (ASM proliferation and extracellular matrix formation) in the context of airway disease. In the present study, we examined the role of BDNF in allergen-induced airway inflammation using 2 transgenic models: 1) tropomyosin-related kinase B (TrkB) conditional knockin (TrkBKI) mice allowing for inducible, reversible disruption of BDNF receptor kinase activity by administration of 1NMPP1, a PP1 derivative, and 2) smooth muscle-specific BDNF knockout (BDNFfl/fl/SMMHC11Cre/0) mice. Adult mice were intranasally challenged with PBS or mixed allergen ( Alternaria alternata, Aspergillus fumigatus, house dust mite, and ovalbumin) for 4 wk. Our data show that administration of 1NMPP1 in TrkBKI mice during the 4-wk allergen challenge blunted airway hyperresponsiveness (AHR) and reduced fibronectin mRNA expression in ASM layers but did not reduce inflammation per se. Smooth muscle-specific deletion of BDNF reduced AHR and blunted airway fibrosis but did not significantly alter airway inflammation. Together, our novel data indicate that TrkB signaling is a key modulator of AHR and that smooth muscle-derived BDNF mediates these effects during allergic airway inflammation.-Britt, R. D., Jr., Thompson, M. A., Wicher, S. A., Manlove, L. J., Roesler, A., Fang, Y.-H., Roos, C., Smith, L., Miller, J. D., Pabelick, C. M., Prakash, Y. S. Smooth muscle brain-derived neurotrophic factor contributes to airway hyperreactivity in a mouse model of allergic asthma.

Pathophysiology of Aortic Valve Stenosis: Is It Both Fibrocalcific and Sex Specific?

Our understanding of the fundamental biology and identification of efficacious therapeutic targets in aortic valve stenosis has lagged far behind the fields of atherosclerosis and heart failure. In this review, we highlight the most clinically relevant problems facing men and women with fibrocalcific aortic valve stenosis, discuss the fundamental biology underlying valve calcification and fibrosis, and identify key molecular points of intersection with sex hormone signaling.

Sex-related differences in calcific aortic stenosis: correlating clinical and echocardiographic characteristics and computed tomography aortic valve calcium score to excised aortic valve weight.

AIMS: Calcific aortic valve stenosis (AS) is purportedly associated with less calcium burden in women than in men. We sought to examine sex-related differences and correlates of surgically excised aortic valve weight (AVW) in pure AS. METHODS AND RESULTS: Clinical and echocardiographic characteristics of 888 consecutive patients who underwent aortic valve replacement for severe AS were correlated to AVW, and in 126 patients, AVW was also correlated to computed tomography aortic valve calcium (AVC) score. Women and men had similar indexed valve area (0.42 ± 0.09 vs. 0.42 ± 0.07 cm (2)/m(2), P = 0.95) and mean systolic gradient (53 ± 15 vs. 52 ± 13 mmHg, P = 0.11), but women had higher New York Heart Association class (2.63 ± 0.70 vs. 2.50 ± 0.70, P = 0.01) and less prevalent coronary artery disease (38 vs. 52%, P < 0.0001). Aortic valve weight was lower in women (1.94 ± 0.88 vs. 3.08 ± 1.32 g, P < 0.0001) even when indexed to body surface area (1.09 ± 0.48 vs. 1.48 ± 0.62 g/m(2), P < 0.0001) or left ventricular outflow tract (LVOT) area (0.54 ± 0.23 vs. 0.71 ± 0.29 g/cm(2), P < 0.0001). Using multivariate analysis, male sex (P < 0.0001), bicuspid valve (P < 0.0001), and larger LVOT area (P < 0.0001) were the major determinants of increased AVW, along with current cigarette smoking (P = 0.007). Diabetes (P = 0.004) and hypertension (P = 0.03) were independently associated with lower AVW. Aortic valve calcium correlated well with AVW (r = 0.81, P < 0.0001) and was lower in women than in men (2520 ± 1199 vs. 3606 ± 1632 arbitrary units, P < 0.0001). CONCLUSIONS: Despite the same degree of AS severity, women have less AVC and lower AVW compared with men, irrespective of valve morphology. Aortic valve calcium is correlated to excised AVW. Hypertension, diabetes, and current cigarette smoking were independently associated with AVW.

Molecular Determinants of Calpain-dependent Cleavage of Junctophilin-2 Protein in Cardiomyocytes.

Junctophilin-2 (JP2), a membrane-binding protein that provides a structural bridge between the plasmalemma and sarcoplasmic reticulum, is essential for precise Ca(2+)-induced Ca(2+) release during excitation-contraction coupling in cardiomyocytes. In animal and human failing hearts, expression of JP2 is decreased markedly, but the molecular mechanisms underlying JP2 down-regulation remain incompletely defined. In mouse hearts, ischemia/reperfusion injury resulted in acute JP2 down-regulation, which was attenuated by pretreatment with the calpain inhibitor MDL-28170 or by transgenic overexpression of calpastatin, an endogenous calpain inhibitor. Using a combination of computational analysis to predict calpain cleavage sites and in vitro calpain proteolysis reactions, we identified four putative calpain cleavage sites within JP2 with three N-terminal and one C-terminal cleavage sites. Mutagenesis defined the C-terminal region of JP2 as the predominant calpain cleavage site. Exogenous expression of putative JP2 cleavage fragments was not sufficient to rescue Ca(2+) handling in JP2-deficient cardiomyocytes, indicating that cleaved JP2 is non-functional for normal Ca(2+)-induced Ca(2+) release. These data provide new molecular insights into the posttranslational regulatory mechanisms of JP2 in cardiac diseases.

Microtubule-mediated defects in junctophilin-2 trafficking contribute to myocyte transverse-tubule remodeling and Ca2+ handling dysfunction in heart failure.

BACKGROUND: Cardiac dysfunction in failing hearts of human patients and animal models is associated with both microtubule densification and transverse-tubule (T-tubule) remodeling. Our objective was to investigate whether microtubule densification contributes to T-tubule remodeling and excitation-contraction coupling dysfunction in heart disease. METHODS AND RESULTS: In a mouse model of pressure overload-induced cardiomyopathy by transaortic banding, colchicine, a microtubule depolymerizer, significantly ameliorated T-tubule remodeling and cardiac dysfunction. In cultured cardiomyocytes, microtubule depolymerization with nocodazole or colchicine profoundly attenuated T-tubule impairment, whereas microtubule polymerization/stabilization with taxol accelerated T-tubule remodeling. In situ immunofluorescence of heart tissue sections demonstrated significant disorganization of junctophilin-2 (JP2), a protein that bridges the T-tubule and sarcoplasmic reticulum membranes, in transaortic banded hearts as well as in human failing hearts, whereas colchicine injection significantly preserved the distribution of JP2 in transaortic banded hearts. In isolated mouse cardiomyocytes, prolonged culture or treatment with taxol resulted in pronounced redistribution of JP2 from T-tubules to the peripheral plasma membrane, without changing total JP2 expression. Nocodazole treatment antagonized JP2 redistribution. Moreover, overexpression of a dominant-negative mutant of kinesin 1, a microtubule motor protein responsible for anterograde trafficking of proteins, protected against JP2 redistribution and T-tubule remodeling in culture. Finally, nocodazole treatment improved Ca(2+) handling in cultured myocytes by increasing the amplitude of Ca(2+) transients and reducing the frequency of Ca(2+) sparks. CONCLUSION: Our data identify a mechanistic link between microtubule densification and T-tubule remodeling and reveal microtubule-mediated JP2 redistribution as a novel mechanism for T-tubule disruption, loss of excitation-contraction coupling, and heart failure.

Reward and Toxicity of Cocaine Metabolites Generated by Cocaine Hydrolase.

Butyrylcholinesterase (BChE) gene therapy is emerging as a promising concept for treatment of cocaine addiction. BChE levels after gene transfer can rise 1000-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. For months or years, gene transfer of a BChE mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal.

Fibrocalcific aortic valve disease: opportunity to understand disease mechanisms using mouse models.

Studies in vitro and in vivo continue to identify complex-regulated mechanisms leading to overt fibrocalcific aortic valve disease (FCAVD). Assessment of the functional impact of those processes requires careful studies of models of FCAVD in vivo. Although the genetic basis for FCAVD is unknown for most patients with FCAVD, several disease-associated genes have been identified in humans and mice. Some gene products which regulate valve development in utero also protect against fibrocalcific disease during postnatal aging. Valve calcification can occur via processes that resemble bone formation. But valve calcification can also occur by nonosteogenic mechanisms, such as formation of calcific apoptotic nodules. Anticalcific interventions might preferentially target either osteogenic or nonosteogenic calcification. Although FCAVD and atherosclerosis share several risk factors and mechanisms, there are fundamental differences between arteries and the aortic valve, with respect to disease mechanisms and responses to therapeutic interventions. Both innate and acquired immunity are likely to contribute to FCAVD. Angiogenesis is a feature of inflammation, but may also contribute independently to progression of FCAVD, possibly by actions of pericytes that are associated with new blood vessels. Several therapeutic interventions seem to be effective in attenuating the development of FCAVD in mice. Therapies which are effective early in the course of FCAVD, however, are not necessarily effective in established disease.

Vascular function during prolonged progression and regression of atherosclerosis in mice.

OBJECTIVE: Endothelial dysfunction is associated with atherosclerosis in mice, but it is difficult to reduce cholesterol levels enough to study regression of atherosclerosis in genetically modified mice. The goal of this study was to examine vascular structure and function before and after reducing elevated plasma lipid levels with a genetic switch in Reversa mice, and identify novel mechanisms contributing to structural and functional improvements in the vasculature after reduction of blood lipids. METHODS AND RESULTS: After 6 months of hypercholesterolemia, endothelial function (maximum relaxation to acetylcholine) in aorta was impaired and responses to nitric oxide were unaffected. Further impairment in endothelial function was observed after 12 months of hypercholesterolemia and was associated with reductions in sensitivity to nitric oxide. Expression of dihydrofolate reductase was reduced at 6 and 12 months, and addition of the tetrahydrobiopterin precursor sepiapterin significantly improved endothelial function. Reducing cholesterol levels at 6 months normalized dihydrofolate reductase expression and prevented further impairment in endothelial function. Similar functional changes were observed after 12 months of hypercholesterolemia followed by 2 months of lipid lowering. CONCLUSIONS: Our data suggest that endothelial dysfunction after prolonged hypercholesterolemia is the result of both impairment of sensitivity to nitric oxide and reduced nitric oxide synthase cofactor bioavailability. Both of these changes can be prevented by normalizing blood lipids during moderately severe or advanced atherosclerosis.

Physical Resilience as a Predictor of Lifespan and Late-Life Health in Genetically Heterogeneous Mice.

Dynamic measures of resilience-the ability to resist and recover from a challenge-may be informative of the rate of aging before overt manifestations such as chronic disease, disability, and frailty. From this perspective mid-life resilience may predict longevity and late-life health. To test this hypothesis, we developed simple, reproducible, clinically relevant challenges, and outcome measures of physical resilience that revealed differences between and within age groups of genetically heterogeneous mice, and then examined associations between mid-life resilience and both lifespan and late-life measures of physiological function. We demonstrate that time to recovery from isoflurane anesthesia and weight change following a regimen of chemotherapy significantly differed among young, middle-aged, and older mice, and were more variable in older mice. Females that recovered faster than the median time from anesthesia (more resilient) at 12 months of age lived 8% longer than their counterparts, while more resilient males in mid-life exhibited better cardiac (fractional shortening and left ventricular volumes) and metabolic (glucose tolerance) function at 24 months of age. Moreover, female mice with less than the median weight loss at Day 3 of the cisplatin challenge lived 8% longer than those that lost more weight. In contrast, females who had more weight loss between Days 15 and 20 were relatively protected against early death. These data suggest that measures of physical resilience in mid-life may provide information about individual differences in aging, lifespan, and key parameters of late-life health.

Transcriptional and phenotypic changes in aorta and aortic valve with aging and MnSOD deficiency in mice.

The purpose of this study was to characterize changes in antioxidant and age-related gene expression in aorta and aortic valve with aging, and test the hypothesis that increased mitochondrial oxidative stress accelerates age-related endothelial and aortic valve dysfunction. Wild-type (MnSOD(+/+)) and manganese SOD heterozygous haploinsufficient (MnSOD(+/-)) mice were studied at 3 and 18 mo of age. In aorta from wild-type mice, antioxidant expression was preserved, although there were age-associated increases in Nox2 expression. Haploinsufficiency of MnSOD did not alter antioxidant expression in aorta, but increased expression of Nox2. When compared with that of aorta, age-associated reductions in antioxidant expression were larger in aortic valves from wild-type and MnSOD haploinsufficient mice, although Nox2 expression was unchanged. Similarly, sirtuin expression was relatively well-preserved in aorta from both genotypes, whereas expression of SIRT1, SIRT2, SIRT3, SIRT4, and SIRT6 were significantly reduced in the aortic valve. Expression of p16(ink4a), a marker of cellular senescence, was profoundly increased in both aorta and aortic valve from MnSOD(+/+) and MnSOD(+/-) mice. Functionally, we observed comparable age-associated reductions in endothelial function in aorta from both MnSOD(+/+) and MnSOD(+/-) mice. Interestingly, inhibition of NAD(P)H oxidase with apocynin or gp91ds-tat improved endothelial function in MnSOD(+/+) mice but significantly impaired endothelial function in MnSOD(+/-) mice at both ages. Aortic valve function was not impaired by aging or MnSOD haploinsufficiency. Changes in antioxidant and sirtuin gene expression with aging differ dramatically between aorta and aortic valve. Furthermore, although MnSOD does not result in overt cardiovascular dysfunction with aging, compensatory transcriptional responses to MnSOD deficiency appear to be tissue specific.

Calcific aortic valve stenosis: methods, models, and mechanisms.

Calcific aortic valve stenosis (CAVS) is a major health problem facing aging societies. The identification of osteoblast-like and osteoclast-like cells in human tissue has led to a major paradigm shift in the field. CAVS was thought to be a passive, degenerative process, whereas now the progression of calcification in CAVS is considered to be actively regulated. Mechanistic studies examining the contributions of true ectopic osteogenesis, nonosseous calcification, and ectopic osteoblast-like cells (that appear to function differently from skeletal osteoblasts) to valvular dysfunction have been facilitated by the development of mouse models of CAVS. Recent studies also suggest that valvular fibrosis, as well as calcification, may play an important role in restricting cusp movement, and CAVS may be more appropriately viewed as a fibrocalcific disease. High-resolution echocardiography and magnetic resonance imaging have emerged as useful tools for testing the efficacy of pharmacological and genetic interventions in vivo. Key studies in humans and animals are reviewed that have shaped current paradigms in the field of CAVS, and suggest promising future areas for research.

Primary mediastinal seminoma presenting with paraneoplastic anti-Hu encephalitis: a case report and literature review.

Primary mediastinal seminomas are exceedingly rare tumors, often localized to the anterior mediastinum. They may present with numerous complications, including superior vena cava syndrome, chylothorax, and pericardial effusions. Less commonly, they may present with paraneoplastic encephalitis. In this report we describe a case of a 19-year-old male with no significant past medical history who presented with bilateral hearing loss, progressive neuropathy, and ataxia. Subsequently the patient was found to have mediastinal mass with a high-titer anti-Hu antibody. To our knowledge, only one other case of mediastinal seminoma presenting with anti-Hu antibodies has been described in the literature. In this report, we describe a rare case of mediastinal seminoma, describe treatment options, and discuss additional known cases presenting with paraneoplastic encephalitis.

Senescent Cells: A Therapeutic Target in Cardiovascular Diseases.

Senescent cell accumulation has been observed in age-associated diseases including cardiovascular diseases. Senescent cells lack proliferative capacity and secrete senescence-associated secretory phenotype (SASP) factors that may cause or worsen many cardiovascular diseases. Therapies targeting senescent cells, especially senolytic drugs that selectively induce senescent cell removal, have been shown to delay, prevent, alleviate, or treat multiple age-associated diseases in preclinical models. Some senolytic clinical trials have already been completed or are underway for a number of diseases and geriatric syndromes. Understanding how cellular senescence affects the various cell types in the cardiovascular system, such as endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells, progenitor cells, and cardiomyocytes, is important to facilitate translation of senotherapeutics into clinical interventions. This review highlights: (1) the characteristics of senescent cells and their involvement in cardiovascular diseases, focusing on the aforementioned cardiovascular cell types, (2) evidence about senolytic drugs and other senotherapeutics, and (3) the future path and clinical potential of senotherapeutics for cardiovascular diseases.

Impact of ACE2 deficiency and oxidative stress on cerebrovascular function with aging.

BACKGROUND AND PURPOSE: Angiotensin II produces oxidative stress and endothelial dysfunction in cerebral arteries, and angiotensin II type I receptors may play a role in longevity and vascular aging. Angiotensin-converting enzyme type 2 (ACE2) converts angiotensin II to angiotensin (1-7) and thus, may protect against effects of angiotensin II. We hypothesized that ACE2 deficiency increases oxidative stress and endothelial dysfunction in cerebral arteries and examined the role of ACE2 in age-related cerebrovascular dysfunction. METHODS: Endothelial function, expression of angiotensin system components, NADPH oxidase subunits, and proinflammatory cytokines were examined in cerebral arteries from adult (12 months old) and old (24 months old) ACE2 knockout (KO) and wild-type (WT) mice. The superoxide scavenger tempol was used to examine the role of oxidative stress on endothelial function. RESULTS: Vasodilatation to acetylcholine was impaired in adult ACE2 KO (24±6% [mean±SE]) compared with WT mice (52±7%; P<0.05). In old mice, vasodilatation to acetylcholine was impaired in WT mice (29±6%) and severely impaired in ACE2 KO mice (7±5%). Tempol improved endothelial function in adult and old ACE2 KO and WT mice. Aging increased mRNA for tumor necrosis factor-α in WT mice, and significantly increased mRNA levels of NAPDH oxidase 2, p47(phox), and Regulator of calcineurin 1 in both ACE2 KO and WT mice. mRNA levels of angiotensin system components did not change during aging. CONCLUSIONS: ACE2 deficiency impaired endothelial function in cerebral arteries from adult mice and augmented endothelial dysfunction during aging. Oxidative stress plays a critical role in cerebrovascular dysfunction induced by ACE2 deficiency and aging.