Knockdown of ANGPTL2 promotes left ventricular systolic dysfunction by upregulation of NOX4 in mice.

Background: Angiopoietin-like 2 (ANGPTL2) is a pro-inflammatory and pro-oxidant circulating protein that predicts and promotes chronic inflammatory diseases such as atherosclerosis in humans. Transgenic murine models demonstrated the deleterious role of ANGPTL2 in vascular diseases, while deletion of ANGPTL2 was protective. The nature of its role in cardiac tissues is, however, less clear. Indeed, in adult mice knocked down (KD) for ANGPTL2, we recently reported a mild left ventricular (LV) dysfunction originating from a congenital aortic valve stenosis, demonstrating that ANGPTL2 is essential to cardiac development and function. Hypothesis: Because we originally demonstrated that the KD of ANGPTL2 protected vascular endothelial function via an upregulation of arterial NOX4, promoting the beneficial production of dilatory H2O2, we tested the hypothesis that increased cardiac NOX4 could negatively affect cardiac redox and remodeling and contribute to LV dysfunction observed in adult Angptl2-KD mice. Methods and results: Cardiac expression and activity of NOX4 were higher in KD mice, promoting higher levels of cardiac H2O2 when compared to wild-type (WT) mice. Immunofluorescence showed that ANGPTL2 and NOX4 were co-expressed in cardiac cells from WT mice and both proteins co-immunoprecipitated in HEK293 cells, suggesting that ANGPTL2 and NOX4 physically interact. Pressure overload induced by transverse aortic constriction surgery (TAC) promoted LV systolic dysfunction in WT mice but did not further exacerbate the dysfunction in KD mice. Importantly, the severity of LV systolic dysfunction in KD mice (TAC and control SHAM) correlated with cardiac Nox4 expression. Injection of an adeno-associated virus (AAV9) delivering shRNA targeting cardiac Nox4 expression fully reversed LV systolic dysfunction in KD-SHAM mice, demonstrating the causal role of NOX4 in cardiac dysfunction in KD mice. Targeting cardiac Nox4 expression in KD mice also induced an antioxidant response characterized by increased expression of NRF2/KEAP1 and catalase. Conclusion: Together, these data reveal that the absence of ANGPTL2 induces an upregulation of cardiac NOX4 that contributes to oxidative stress and LV dysfunction. By interacting and repressing cardiac NOX4, ANGPTL2 could play a new beneficial role in the maintenance of cardiac redox homeostasis and function.

Mechanisms of Vascular Inflammation and Potential Therapeutic Targets: A Position Paper From the ESH Working Group on Small Arteries.

Inflammatory responses in small vessels play an important role in the development of cardiovascular diseases, including hypertension, stroke, and small vessel disease. This involves various complex molecular processes including oxidative stress, inflammasome activation, immune-mediated responses, and protein misfolding, which together contribute to microvascular damage. In addition, epigenetic factors, including DNA methylation, histone modifications, and microRNAs influence vascular inflammation and injury. These phenomena may be acquired during the aging process or due to environmental factors. Activation of proinflammatory signaling pathways and molecular events induce low-grade and chronic inflammation with consequent cardiovascular damage. Identifying mechanism-specific targets might provide opportunities in the development of novel therapeutic approaches. Monoclonal antibodies targeting inflammatory cytokines and epigenetic drugs, show promise in reducing microvascular inflammation and associated cardiovascular diseases. In this article, we provide a comprehensive discussion of the complex mechanisms underlying microvascular inflammation and offer insights into innovative therapeutic strategies that may ameliorate vascular injury in cardiovascular disease.

SEC31A may be associated with pituitary hormone deficiency and gonadal dysgenesis.

PURPOSE: Disorders/differences of sex development (DSD) result from variants in many different human genes but, frequently, have no detectable molecular cause. METHODS: Detailed clinical and genetic phenotyping was conducted on a family with three children. A Sec31a animal model and functional studies were used to investigate the significance of the findings. RESULTS: By trio whole-exome DNA sequencing we detected a heterozygous de novo nonsense SEC31A variant, in three children of healthy non-consanguineous parents. The children had different combinations of disorders that included complete gonadal dysgenesis and multiple pituitary hormone deficiency. SEC31A encodes a component of the COPII coat protein complex, necessary for intracellular anterograde vesicle-mediated transport between the endoplasmic reticulum (ER) and Golgi. CRISPR-Cas9 targeted knockout of the orthologous Sec31a gene region resulted in early embryonic lethality in homozygous mice. mRNA expression of ER-stress genes ATF4 and CHOP was increased in the children, suggesting defective protein transport. The pLI score of the gene, from gnomAD data, is 0.02. CONCLUSIONS: SEC31A might underlie a previously unrecognised clinical syndrome comprising gonadal dysgenesis, multiple pituitary hormone deficiencies, dysmorphic features and developmental delay. However, a variant that remains undetected, in a different gene, may alternatively be causal in this family.

Effects of Sex Hormones on Vascular Reactivity in Boys With Hypospadias.

BACKGROUND: Arteries from boys with hypospadias demonstrate hypercontractility and impaired vasorelaxation. The role of sex hormones in these responses in unclear. AIMS: We compared effects of sex steroids on vascular reactivity in healthy boys and boys with hypospadias. METHODS: Excess foreskin tissue was obtained from 11 boys undergoing hypospadias repair (cases) and 12 undergoing routine circumcision (controls) (median age [range], 1.5 [1.2-2.7] years) and small resistance arteries were isolated. Vessels were mounted on wire myographs and vascular reactivity was assessed in the absence/presence of 17ß-estradiol, dihydrotestosterone (DHT), and testosterone. RESULTS: In controls, testosterone and 17ß-estradiol increased contraction (percent of maximum contraction [Emax]: 83.74 basal vs 125.4 after testosterone, P < .0002; and 83.74 vs 110.2 after estradiol, P = .02). 17ß-estradiol reduced vasorelaxation in arteries from controls (Emax: 10.6 vs 15.6 to acetylcholine, P < .0001; and Emax: 14.6 vs 20.5 to sodium nitroprusside, P < .0001). In hypospadias, testosterone (Emax: 137.9 vs 107.2, P = .01) and 17ß-estradiol (Emax: 156.9 vs 23.6, P < .0001) reduced contraction. Androgens, but not 17ß-estradiol, increased endothelium-dependent and endothelium-independent vasorelaxation in cases (Emax: 77.3 vs 51.7 with testosterone, P = .02; and vs 48.2 with DHT to acetylcholine, P = .0001; Emax: 43.0 vs 39.5 with testosterone, P = .02; and 39.6 vs 37.5 with DHT to sodium nitroprusside, P = .04). CONCLUSION: In healthy boys, testosterone and 17ß-estradiol promote a vasoconstrictor phenotype, whereas in boys with hypospadias, these sex hormones reduce vasoconstriction, with androgens promoting vasorelaxation. Differences in baseline artery function may therefore be sex hormone-independent and the impact of early-life variations in androgen exposure on vascular function needs further study.

Recent Advances in Understanding the Mechanistic Role of Transient Receptor Potential Ion Channels in Patients With Hypertension.

The transient receptor potential (TRP) channel superfamily is a group of nonselective cation channels that function as cellular sensors for a wide range of physical, chemical, and environmental stimuli. According to sequence homology, TRP channels are categorized into 6 subfamilies: TRP canonical, TRP vanilloid, TRP melastatin, TRP ankyrin, TRP mucolipin, and TRP polycystin. They are widely expressed in different cell types and tissues and have essential roles in various physiological and pathological processes by regulating the concentration of ions (Ca2+, Mg2+, Na+, and K+) and influencing intracellular signalling pathways. Human data and experimental models indicate the importance of TRP channels in vascular homeostasis and hypertension. Furthermore, TRP channels have emerged as key players in oxidative stress and inflammation, important in the pathophysiology of cardiovascular diseases, including hypertension. In this review, we present an overview of the TRP channels with a focus on their role in hypertension. In particular, we highlight mechanisms activated by TRP channels in vascular smooth muscle and endothelial cells and discuss their contribution to processes underlying vascular dysfunction in hypertension.

Oxidative Stress and Endoplasmic Reticular Stress Interplay in the Vasculopathy of Hypertension.

Under physiologic conditions, reactive oxygen species (ROS) function as signalling molecules that control cell function. However, in pathologic conditions, increased generation of ROS triggers oxidative stress, which plays a role in vascular changes associated with hypertension, including endothelial dysfunction, vascular reactivity, and arterial remodelling (termed the vasculopathy of hypertension). The major source of ROS in the vascular system is NADPH oxidase (NOX). Increased NOX activity drives vascular oxidative stress in hypertension. Molecular mechanisms underlying vascular damage in hypertension include activation of redox-sensitive signalling pathways, post-translational modification of proteins, and oxidative damage of DNA and cytoplasmic proteins. In addition, oxidative stress leads to accumulation of proteins in the endoplasmic reticulum (ER) (termed ER stress), with consequent activation of the unfolded protein response (UPR). ER stress is emerging as a potential player in hypertension as abnormal protein folding in the ER leads to oxidative stress and dysregulated activation of the UPR promotes inflammation and injury in vascular and cardiac cells. In addition, the ER engages in crosstalk with exogenous sources of ROS, such as mitochondria and NOX, which can amplify redox processes. Here we provide an update of the role of ROS and NOX in hypertension and discuss novel concepts on the interplay between oxidative stress and ER stress.

SARS-CoV-2 spike protein induces endothelial inflammation via ACE2 independently of viral replication.

COVID-19, caused by SARS-CoV-2, is a respiratory disease associated with inflammation and endotheliitis. Mechanisms underling inflammatory processes are unclear, but angiotensin converting enzyme 2 (ACE2), the receptor which binds the spike protein of SARS-CoV-2 may be important. Here we investigated whether spike protein binding to ACE2 induces inflammation in endothelial cells and determined the role of ACE2 in this process. Human endothelial cells were exposed to SARS-CoV-2 spike protein, S1 subunit (rS1p) and pro-inflammatory signaling and inflammatory mediators assessed. ACE2 was modulated pharmacologically and by siRNA. Endothelial cells were also exposed to SARS-CoV-2. rSP1 increased production of IL-6, MCP-1, ICAM-1 and PAI-1, and induced NFkB activation via ACE2 in endothelial cells. rS1p increased microparticle formation, a functional marker of endothelial injury. ACE2 interacting proteins involved in inflammation and RNA biology were identified in rS1p-treated cells. Neither ACE2 expression nor ACE2 enzymatic function were affected by rSP1. Endothelial cells exposed to SARS-CoV-2 virus did not exhibit viral replication. We demonstrate that rSP1 induces endothelial inflammation via ACE2 through processes that are independent of ACE2 enzymatic activity and viral replication. We define a novel role for ACE2 in COVID-19- associated endotheliitis.

Impact of Environmental Factors on Hypertension and Associated Cardiovascular Disease.

Hypertension is the primary cause of cardiovascular diseases and is responsible for nearly 9 million deaths worldwide annually. Increasing evidence indicates that in addition to pathophysiologic processes, numerous environmental factors, such as geographic location, lifestyle choices, socioeconomic status, and cultural practices, influence the risk, progression, and severity of hypertension, even in the absence of genetic risk factors. In this review, we discuss the impact of some environmental determinants on hypertension. We focus on clinical data from large population studies and discuss some potential molecular and cellular mechanisms. We highlight how these environmental determinants are interconnected, as small changes in one factor might affect others, and further affect cardiovascular health. In addition, we discuss the crucial impact of socioeconomic factors and how these determinants influence diverse communities with economic disparities. Finally, we address opportunities and challenges for new research to address gaps in knowledge on understanding molecular mechanisms whereby environmental factors influence development of hypertension and associated cardiovascular disease.

The importance of microvascular inflammation in ageing and age-related diseases: a position paper from the ESH working group on small arteries, section of microvascular inflammation.

Microcirculation is pervasive and orchestrates a profound regulatory cross-talk with the surrounding tissue and organs. Similarly, it is one of the earliest biological systems targeted by environmental stressors and consequently involved in the development and progression of ageing and age-related disease. Microvascular dysfunction, if not targeted, leads to a steady derangement of the phenotype, which cumulates comorbidities and eventually results in a nonrescuable, very high-cardiovascular risk. Along the broad spectrum of pathologies, both shared and distinct molecular pathways and pathophysiological alteration are involved in the disruption of microvascular homeostasis, all pointing to microvascular inflammation as the putative primary culprit. This position paper explores the presence and the detrimental contribution of microvascular inflammation across the whole spectrum of chronic age-related diseases, which characterise the 21st-century healthcare landscape. The manuscript aims to strongly affirm the centrality of microvascular inflammation by recapitulating the current evidence and providing a clear synoptic view of the whole cardiometabolic derangement. Indeed, there is an urgent need for further mechanistic exploration to identify clear, very early or disease-specific molecular targets to provide an effective therapeutic strategy against the otherwise unstoppable rising prevalence of age-related diseases.

Vasoprotective effects of NOX4 are mediated via polymerase and transient receptor potential melastatin 2 cation channels in endothelial cells.

BACKGROUND: NOX4 activation has been implicated to have vasoprotective and blood pressure (BP)-lowering effects. Molecular mechanisms underlying this are unclear, but NOX4-induced regulation of the redox-sensitive Ca 2+ channel TRPM2 and effects on endothelial nitric oxide synthase (eNOS)-nitric oxide signalling may be important. METHOD: Wild-type and LinA3, renin-expressing hypertensive mice, were crossed with NOX4 knockout mice. Vascular function was measured by myography. Generation of superoxide (O 2- ) and hydrogen peroxide (H 2 O 2 ) were assessed by lucigenin and amplex red, respectively, and Ca 2+ influx by Cal-520 fluorescence in rat aortic endothelial cells (RAEC). RESULTS: BP was increased in NOX4KO, LinA3 and LinA3/NOX4KO mice. This was associated with endothelial dysfunction and vascular remodelling, with exaggerated effects in NOX4KO groups. The TRPM2 activator, ADPR, improved vascular relaxation in LinA3/NOX4KO mice, an effect recapitulated by H 2 O 2 . Inhibition of PARP and TRPM2 with olaparib and 2-APB, respectively, recapitulated endothelial dysfunction in NOX4KO. In endothelial cells, Ang II increased H 2 O 2 generation and Ca 2+ influx, effects reduced by TRPM2 siRNA, TRPM2 inhibitors (8-br-cADPR, 2-APB), olaparib and GKT137831 (NOX4 inhibitor). Ang II-induced eNOS activation was blocked by NOX4 and TRPM2 siRNA, GKT137831, PEG-catalase and 8-br-cADPR. CONCLUSION: Our findings indicate that NOX4-induced H 2 O 2 production activates PARP/TRPM2, Ca 2+ influx, eNOS activation and nitric oxide release in endothelial cells. NOX4 deficiency impairs Ca 2+ homeostasis leading to endothelial dysfunction, an effect exacerbated in hypertension. We define a novel pathway linking endothelial NOX4/H 2 O 2 to eNOS/nitric oxide through PARP/TRPM2/Ca 2+ . This vasoprotective pathway is perturbed when NOX4 is downregulated and may have significance in conditions associated with endothelial dysfunction, including hypertension.

Notch3/Hes5 Induces Vascular Dysfunction in Hypoxia-Induced Pulmonary Hypertension Through ER Stress and Redox-Sensitive Pathways.

BACKGROUND: Notch3 (neurogenic locus notch homolog protein 3) is implicated in vascular diseases, including pulmonary hypertension (PH)/pulmonary arterial hypertension. However, molecular mechanisms remain elusive. We hypothesized increased Notch3 activation induces oxidative and endoplasmic reticulum (ER) stress and downstream redox signaling, associated with procontractile pulmonary artery state, pulmonary vascular dysfunction, and PH development. METHODS: Studies were performed in TgNotch3R169C mice (harboring gain-of-function [GOF] Notch3 mutation) exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in pulmonary artery smooth muscle cells from pulmonary arterial hypertension patients and in mouse lung. Notch3-regulated genes/proteins, ER stress, ROCK (Rho-associated kinase) expression/activity, Ca2+ transients and generation of reactive oxygen species, and nitric oxide were measured. Pulmonary vascular reactivity was assessed in the presence of fasudil (ROCK inhibitor) and 4-phenylbutyric acid (ER stress inhibitor). RESULTS: Hypoxia induced a more severe PH phenotype in TgNotch3R169C mice versus controls. TgNotch3R169C mice exhibited enhanced Notch3 activation and expression of Notch3 targets Hes Family BHLH Transcription Factor 5 (Hes5), with increased vascular contraction and impaired vasorelaxation that improved with fasudil/4-phenylbutyric acid. Notch3 mutation was associated with increased pulmonary vessel Ca2+ transients, ROCK activation, ER stress, and increased reactive oxygen species generation, with reduced NO generation and blunted sGC (soluble guanylyl cyclase)/cGMP signaling. These effects were ameliorated by N-acetylcysteine. pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension recapitulated Notch3/Hes5 signaling, ER stress and redox changes observed in PH mice. CONCLUSIONS: Notch3 GOF amplifies vascular dysfunction in hypoxic PH. This involves oxidative and ER stress, and ROCK. We highlight a novel role for Notch3/Hes5-redox signaling and important interplay between ER and oxidative stress in PH.

Vascular mechanisms of post-COVID-19 conditions: Rho-kinase is a novel target for therapy.

BACKGROUND: In post-coronavirus disease-19 (post-COVID-19) conditions (long COVID), systemic vascular dysfunction is implicated, but the mechanisms are uncertain, and the treatment is imprecise. METHODS AND RESULTS: Patients convalescing after hospitalization for COVID-19 and risk factor matched controls underwent multisystem phenotyping using blood biomarkers, cardiorenal and pulmonary imaging, and gluteal subcutaneous biopsy (NCT04403607). Small resistance arteries were isolated and examined using wire myography, histopathology, immunohistochemistry, and spatial transcriptomics. Endothelium-independent (sodium nitroprusside) and -dependent (acetylcholine) vasorelaxation and vasoconstriction to the thromboxane A2 receptor agonist, U46619, and endothelin-1 (ET-1) in the presence or absence of a RhoA/Rho-kinase inhibitor (fasudil), were investigated. Thirty-seven patients, including 27 (mean age 57 years, 48% women, 41% cardiovascular disease) 3 months post-COVID-19 and 10 controls (mean age 57 years, 20% women, 30% cardiovascular disease), were included. Compared with control responses, U46619-induced constriction was increased (P = 0.002) and endothelium-independent vasorelaxation was reduced in arteries from COVID-19 patients (P < 0.001). This difference was abolished by fasudil. Histopathology revealed greater collagen abundance in COVID-19 arteries {Masson's trichrome (MT) 69.7% [95% confidence interval (CI): 67.8-71.7]; picrosirius red 68.6% [95% CI: 64.4-72.8]} vs. controls [MT 64.9% (95% CI: 59.4-70.3) (P = 0.028); picrosirius red 60.1% (95% CI: 55.4-64.8), (P = 0.029)]. Greater phosphorylated myosin light chain antibody-positive staining in vascular smooth muscle cells was observed in COVID-19 arteries (40.1%; 95% CI: 30.9-49.3) vs. controls (10.0%; 95% CI: 4.4-15.6) (P < 0.001). In proof-of-concept studies, gene pathways associated with extracellular matrix alteration, proteoglycan synthesis, and viral mRNA replication appeared to be upregulated. CONCLUSION: Patients with post-COVID-19 conditions have enhanced vascular fibrosis and myosin light change phosphorylation. Rho-kinase activation represents a novel therapeutic target for clinical trials.

Role of PARP and TRPM2 in VEGF Inhibitor-Induced Vascular Dysfunction.

Background Hypertension and vascular toxicity are major unwanted side effects of antiangiogenic drugs, such as vascular endothelial growth factor inhibitors (VEGFis), which are effective anticancer drugs but have unwanted side effects, including vascular toxicity and hypertension. Poly (ADP-ribose) polymerase (PARP) inhibitors, used to treat ovarian and other cancers, have also been associated with elevated blood pressure. However, when patients with cancer receive both olaparib, a PARP inhibitor, and VEGFi, the risk of blood pressure elevation is reduced. Underlying molecular mechanisms are unclear, but PARP-regulated transient receptor potential cation channel, subfamily M, member 2 (TRPM2), a redox-sensitive calcium channel, may be important. We investigated whether PARP/TRPM2 plays a role in VEGFi-induced vascular dysfunction and whether PARP inhibition ameliorates the vasculopathy associated with VEGF inhibition. Methods and Results Human vascular smooth muscle cells (VSMCs), human aortic endothelial cells, and wild-type mouse mesenteric arteries were studied. Cells/arteries were exposed to axitinib (VEGFi) alone and in combination with olaparib. Reactive oxygen species production, Ca2+ influx, protein/gene analysis, PARP activity, and TRPM2 signaling were assessed in VSMCs, and nitric oxide levels were determined in endothelial cells. Vascular function was assessed by myography. Axitinib increased PARP activity in VSMCs in a reactive oxygen species-dependent manner. Endothelial dysfunction and hypercontractile responses were ameliorated by olaparib and a TRPM2 blocker (8-Br-cADPR). VSMC reactive oxygen species production, Ca2+ influx, and phosphorylation of myosin light chain 20 and endothelial nitric oxide synthase (Thr495) were augmented by axitinib and attenuated by olaparib and TRPM2 inhibition. Proinflammatory markers were upregulated in axitinib-stimulated VSMCs, which was reduced by reactive oxygen species scavengers and PARP-TRPM2 inhibition. Human aortic endothelial cells exposed to combined olaparib and axitinib showed nitric oxide levels similar to VEGF-stimulated cells. Conclusions Axitinib-mediated vascular dysfunction involves PARP and TRPM2, which, when inhibited, ameliorate the injurious effects of VEGFi. Our findings define a potential mechanism whereby PARP inhibitor may attenuate vascular toxicity in VEGFi-treated patients with cancer.

Exosomes and the cardiovascular system: role in cardiovascular health and disease.

Exosomes, which are membrane-bound extracellular vesicles (EVs), are generated in the endosomal compartment of almost all eukaryotic cells. They are formed upon the fusion of multivesicular bodies and the plasma membrane and carry proteins, nucleic acids, lipids and other cellular constituents from their parent cells. Multiple factors influence their production including cell stress and injury, humoral factors, circulating toxins, and oxidative stress. They play an important role in intercellular communication, through their ability to transfer their cargo (proteins, lipids, RNAs) from one cell to another. Exosomes have been implicated in the pathophysiology of various diseases including cardiovascular disease (CVD), cancer, kidney disease, and inflammatory conditions. In addition, circulating exosomes may act as biomarkers for diagnostic and prognostic strategies for several pathological processes. In particular exosome-containing miRNAs have been suggested as biomarkers for the diagnosis and prognosis of myocardial injury, stroke and endothelial dysfunction. They may also have therapeutic potential, acting as vectors to deliver therapies in a targeted manner, such as the delivery of protective miRNAs. Transfection techniques are in development to load exosomes with desired cargo, such as proteins or miRNAs, to achieve up-regulation in the host cell or tissue. These advances in the field have the potential to assist in the detection and monitoring progress of a disease in patients during its early clinical stages, as well as targeted drug delivery.

TRPM7 deficiency exacerbates cardiovascular and renal damage induced by aldosterone-salt.

Hyperaldosteronism causes cardiovascular disease as well as hypomagnesemia. Mechanisms are ill-defined but dysregulation of TRPM7, a Mg2+-permeable channel/α-kinase, may be important. We examined the role of TRPM7 in aldosterone-dependent cardiovascular and renal injury by studying aldosterone-salt treated TRPM7-deficient (TRPM7+/Δkinase) mice. Plasma/tissue [Mg2+] and TRPM7 phosphorylation were reduced in vehicle-treated TRPM7+/Δkinase mice, effects recapitulated in aldosterone-salt-treated wild-type mice. Aldosterone-salt treatment exaggerated vascular dysfunction and amplified cardiovascular and renal fibrosis, with associated increased blood pressure in TRPM7+/Δkinase mice. Tissue expression of Mg2+-regulated phosphatases (PPM1A, PTEN) was downregulated and phosphorylation of Smad3, ERK1/2, and Stat1 was upregulated in aldosterone-salt TRPM7-deficient mice. Aldosterone-induced phosphorylation of pro-fibrotic signaling was increased in TRPM7+/Δkinase fibroblasts, effects ameliorated by Mg2+ supplementation. TRPM7 deficiency amplifies aldosterone-salt-induced cardiovascular remodeling and damage. We identify TRPM7 downregulation and associated hypomagnesemia as putative molecular mechanisms underlying deleterious cardiovascular and renal effects of hyperaldosteronism.

Vascular dysfunction and increased cardiovascular risk in hypospadias.

AIMS: Hypogonadism is associated with cardiovascular disease. However, the cardiovascular impact of hypogonadism during development is unknown. Using hypospadias as a surrogate of hypogonadism, we investigated whether hypospadias is associated with vascular dysfunction and is a risk factor for cardiovascular disease. METHODS AND RESULTS: Our human study spanned molecular mechanistic to epidemiological investigations. Clinical vascular phenotyping was performed in adolescents with hypospadias and controls. Small subcutaneous arteries from penile skin from boys undergoing hypospadias repair and controls were isolated and functional studies were assessed by myography. Vascular smooth muscle cells were used to assess: Rho kinase, reactive oxygen species (ROS), nitric oxide synthase/nitric oxide, and DNA damage. Systemic oxidative stress was assessed in plasma and urine. Hospital episode data compared men with a history of hypospadias vs. controls. In adolescents with hypospadias, systolic blood pressure (P = 0.005), pulse pressure (P = 0.03), and carotid intima-media thickness standard deviation scores (P = 0.01) were increased. Arteries from boys with hypospadias demonstrated increased U46619-induced vasoconstriction (P = 0.009) and reduced acetylcholine-induced endothelium-dependent (P < 0.0001) and sodium nitroprusside-induced endothelium-independent vasorelaxation (P < 0.0001). Men born with hypospadias were at increased risk of arrhythmia [odds ratio (OR) 2.8, 95% confidence interval (CI) 1.4-5.6, P = 0.003]; hypertension (OR 4.2, 95% CI 1.5-11.9, P = 0.04); and heart failure (OR 1.9, 95% CI 1.7-114.3, P = 0.02). CONCLUSION: Hypospadias is associated with vascular dysfunction and predisposes to hypertension and cardiovascular disease in adulthood. Underlying mechanisms involve perturbed Rho kinase- and Nox5/ROS-dependent signalling. Our novel findings delineate molecular mechanisms of vascular injury in hypogonadism, and identify hypospadias as a cardiovascular risk factor in males.

Differential effects of cyclo-oxygenase 1 and 2 inhibition on angiogenesis inhibitor-induced hypertension and kidney damage.

Vascular endothelial growth factor antagonism with angiogenesis inhibitors in cancer patients induces a 'preeclampsia-like' syndrome including hypertension, proteinuria and elevated endothelin (ET)-1. Cyclo-oxygenase (COX) inhibition with aspirin is known to prevent the onset of preeclampsia in high-risk patients. In the present study, we hypothesised that treatment with aspirin would prevent the development of angiogenesis inhibitor-induced hypertension and kidney damage. Our aims were to compare the effects of low-dose (COX-1 inhibition) and high-dose (dual COX-1 and COX-2 inhibition) aspirin on blood pressure, vascular function, oxidative stress, ET-1 and prostanoid levels and kidney damage during angiogenesis-inhibitor therapy in rodents. To this end, Wistar Kyoto rats were treated with vehicle, angiogenesis inhibitor (sunitinib) alone or in combination with low- or high-dose aspirin for 8 days (n=5-7/group). Our results demonstrated that prostacyclin (PGI2) and ET-1 were increased during angiogenesis-inhibitor therapy, while thromboxane (TXA2) was unchanged. Both low- and high-dose aspirin blunted angiogenesis inhibitor-induced hypertension and vascular superoxide production to a similar extent, whereas only high-dose aspirin prevented albuminuria. While circulating TXA2 and prostaglandin F2α levels were reduced by both low- and high-dose aspirin, circulating and urinary levels PGI2 were only reduced by high-dose aspirin. Lastly, treatment with aspirin did not significantly affect ET-1 or vascular function. Collectively our findings suggest that prostanoids contribute to the development of angiogenesis inhibitor-induced hypertension and renal damage and that targeting the prostanoid pathway could be an effective strategy to mitigate the unwanted cardiovascular and renal toxicities associated with angiogenesis inhibitors.

Interferon-stimulated gene 15 pathway is a novel mediator of endothelial dysfunction and aneurysms development in angiotensin II infused mice through increased oxidative stress.

AIMS: Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defence response to microbial infection; however, its contribution to vascular damage associated with hypertension is unknown. METHODS AND RESULTS: Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensin II (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodelling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodelling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation, and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodelling. CONCLUSION: ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.

Central role of c-Src in NOX5- mediated redox signalling in vascular smooth muscle cells in human hypertension.

AIMS: NOX-derived reactive oxygen species (ROS) are mediators of signalling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here, we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction. METHODS AND RESULTS: VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signalling molecules, and actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were down-regulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs, NOX5 was up-regulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1), and hyperphosphorylation (c-Src, PKC, ERK1/2, MLC20) of signalling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2. CONCLUSION: We define NOX5/ROS/c-Src as a novel feedforward signalling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury.

Sex steroids receptors, hypertension, and vascular ageing.

Sex hormone receptors are expressed throughout the vasculature and play an important role in the modulation of blood pressure in health and disease. The functions of these receptors may be important in the understanding of sexual dimorphism observed in the pathophysiology of both hypertension and vascular ageing. The interconnectivity of these factors can be exemplified in postmenopausal females, who with age and estrogen deprivation, surpass males with regard to hypertension prevalence, despite experiencing significantly less disease burden in their estrogen replete youth. Estrogen and androgen receptors mediate their actions via direct genomic effects or rapid non-genomic signaling, involving a host of mediators. The expression and subtype composition of these receptors changes through the lifespan in response to age, disease and hormonal exposure. These factors may promote sex steroid receptor-mediated alterations to the Renin-Angiotensin-Aldosterone System (RAAS), and increases in oxidative stress and inflammation, thereby contributing to the development of hypertension and vascular injury with age.