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.

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.

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.

Lysophosphatidylcholine induces oxidative stress in human endothelial cells via NOX5 activation - implications in atherosclerosis.

OBJECTIVE: The mechanisms involved in NOX5 activation in atherosclerotic processes are not completely understood. The present study tested the hypothesis that lysophosphatidylcholine (LPC), a proatherogenic component of oxLDL, induces endothelial calcium influx, which drives NOX5-dependent reactive oxygen species (ROS) production, oxidative stress, and endothelial cell dysfunction. APPROACH: Human aortic endothelial cells (HAEC) were stimulated with LPC (10-5 M, for different time points). Pharmacological inhibition of NOX5 (Melittin, 10-7 M) and NOX5 gene silencing (siRNA) was used to determine the role of NOX5-dependent ROS production in endothelial oxidative stress induced by LPC. ROS production was determined by lucigenin assay and electron paramagnetic spectroscopy (EPR), calcium transients by Fluo4 fluorimetry, and NOX5 activity and protein expression by pharmacological assays and immunoblotting, respectively. RESULTS: LPC increased ROS generation in endothelial cells at short (15 min) and long (4 h) stimulation times. LPC-induced ROS was abolished by a selective NOX5 inhibitor and by NOX5 siRNA. NOX1/4 dual inhibition and selective NOX1 inhibition only decreased ROS generation at 4 h. LPC increased HAEC intracellular calcium, important for NOX5 activation, and this was blocked by nifedipine and thapsigargin. Bapta-AM, selective Ca2+ chelator, prevented LPC-induced ROS production. NOX5 knockdown decreased LPC-induced ICAM-1 mRNA expression and monocyte adhesion to endothelial cells. CONCLUSION: These results suggest that NOX5, by mechanisms linked to increased intracellular calcium, is key to early LPC-induced endothelial oxidative stress and pro-inflammatory processes. Since these are essential events in the formation and progression of atherosclerotic lesions, the present study highlights an important role for NOX5 in atherosclerosis.

Selective Inhibition of the C-Domain of ACE (Angiotensin-Converting Enzyme) Combined With Inhibition of NEP (Neprilysin): A Potential New Therapy for Hypertension.

[Figure: see text].

Hypertension and Prohypertensive Antineoplastic Therapies in Cancer Patients.

The development of a wide range of novel antineoplastic therapies has improved the prognosis for patients with a wide range of malignancies, which has increased the number of cancer survivors substantially. Despite the oncological benefit, cancer survivors are exposed to short- and long-term adverse cardiovascular toxicities associated with anticancer therapies. Systemic hypertension, the most common comorbidity among cancer patients, is a major contributor to the increased risk for developing these adverse cardiovascular events. Cancer and hypertension have common risk factors, have overlapping pathophysiological mechanisms and hypertension may also be a risk factor for some tumor types. Many cancer therapies have prohypertensive effects. Although some of the mechanisms by which these antineoplastic agents lead to hypertension have been characterized, further preclinical and clinical studies are required to investigate the exact pathophysiology and the optimal management of hypertension associated with anticancer therapy. In this way, monitoring and management of hypertension before, during, and after cancer treatment can be improved to minimize cardiovascular risks. This is vital to optimize cardiovascular health in patients with cancer and survivors, and to ensure that advances in terms of cancer survivorship do not come at the expense of increased cardiovascular toxicities.

Peripheral arteriopathy caused by Notch3 gain-of-function mutation involves ER and oxidative stress and blunting of NO/sGC/cGMP pathway.

Notch3 mutations cause Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), which predisposes to stroke and dementia. CADASIL is characterised by vascular dysfunction and granular osmiophilic material (GOM) accumulation in cerebral small vessels. Systemic vessels may also be impacted by Notch3 mutations. However vascular characteristics and pathophysiological processes remain elusive. We investigated mechanisms underlying the peripheral vasculopathy mediated by CADASIL-causing Notch3 gain-of-function mutation. We studied: (i) small arteries and vascular smooth muscle cells (VSMCs) from TgNotch3R169C mice (CADASIL model), (ii) VSMCs from peripheral arteries from CADASIL patients, and (iii) post-mortem brains from CADASIL individuals. TgNotch3R169C vessels exhibited GOM deposits, increased vasoreactivity and impaired vasorelaxation. Hypercontractile responses were normalised by fasudil (Rho kinase inhibitor) and 4-phenylbutyrate (4-PBA; endoplasmic-reticulum (ER) stress inhibitor). Ca2+ transients and Ca2+ channel expression were increased in CADASIL VSMCs, with increased expression of Rho guanine nucleotide-exchange factors (GEFs) and ER stress proteins. Vasorelaxation mechanisms were impaired in CADASIL, evidenced by decreased endothelial nitric oxide synthase (eNOS) phosphorylation and reduced cyclic guanosine 3',5'-monophosphate (cGMP) levels, with associated increased soluble guanylate cyclase (sGC) oxidation, decreased sGC activity and reduced levels of the vasodilator hydrogen peroxide (H2O2). In VSMCs from CADASIL patients, sGC oxidation was increased and cGMP levels decreased, effects normalised by fasudil and 4-PBA. Cerebral vessels in CADASIL patients exhibited significant oxidative damage. In conclusion, peripheral vascular dysfunction in CADASIL is associated with altered Ca2+ homoeostasis, oxidative stress and blunted eNOS/sGC/cGMP signaling, processes involving Rho kinase and ER stress. We identify novel pathways underlying the peripheral arteriopathy induced by Notch3 gain-of-function mutation, phenomena that may also be important in cerebral vessels.

Comprehensive Characterization of the Vascular Effects of Cisplatin-Based Chemotherapy in Patients With Testicular Cancer.

BACKGROUND: Cisplatin-based chemotherapy increases the risk of cardiovascular and renal disease. OBJECTIVES: We aimed to define the time course, pathophysiology, and approaches to prevent cardiovascular disease associated with cisplatin-based chemotherapy. METHODS: Two cohorts of patients with a history of testicular cancer (n = 53) were recruited. Cohort 1 consisted of 27 men undergoing treatment with: 1) surveillance; 2) 1 to 2 cycles of bleomycin, etoposide, and cisplatin (BEP) chemotherapy (low-intensity cisplatin); or 3) 3 to 4 cycles of BEP (high-intensity cisplatin). Endothelial function (percentage flow-mediated dilatation) and cardiovascular biomarkers were assessed at 6 visits over 9 months. Cohort 2 consisted of 26 men previously treated 1 to 7 years ago with surveillance or 3 to 4 cycles BEP. Vasomotor and fibrinolytic responses to bradykinin, acetylcholine, and sodium nitroprusside were evaluated using forearm venous occlusion plethysmography. RESULTS: In cohort 1, the percentage flow-mediated dilatation decreased 24 h after the first cisplatin dose in patients managed with 3 to 4 cycles BEP (10.9 ± 0.9 vs. 16.7 ± 1.6; p < 0.01) but was unchanged from baseline thereafter. Six weeks after starting 3 to 4 cycles BEP, there were increased serum cholesterol levels (7.2 ± 0.5 mmol/l vs. 5.5 ± 0.2 mmol/l; p = 0.01), hemoglobin A1c (41.8 ± 2.0 mmol/l vs. 35.5 ± 1.2 mmol/l; p < 0.001), von Willebrand factor antigen (62.4 ± 5.4 mmol/l vs. 45.2 ± 2.8 mmol/l; p = 0.048) and cystatin C (0.91 ± 0.07 mmol/l vs. 0.65 ± 0.09 mmol/l; p < 0.01). In cohort 2, intra-arterial bradykinin, acetylcholine, and sodium nitroprusside caused dose-dependent vasodilation (p < 0.0001). Vasomotor responses, endogenous fibrinolytic factor release, and cardiovascular biomarkers were not different in patients managed with 3 to 4 cycles of BEP versus surveillance. CONCLUSIONS: Cisplatin-based chemotherapy induces acute and transient endothelial dysfunction, dyslipidemia, hyperglycemia, and nephrotoxicity in the early phases of treatment. Cardiovascular and renal protective strategies should target the early perichemotherapy period. (Clinical Characterisation of the Vascular Effects of Cis-platinum Based Chemotherapy in Patients With Testicular Cancer [VECTOR], NCT03557177; Intermediate and Long Term Vascular Effects of Cisplatin in Patients With Testicular Cancer [INTELLECT], NCT03557164).

Vascular toxicity associated with anti-angiogenic drugs.

Over the past two decades, the treatment of cancer has been revolutionised by the highly successful introduction of novel molecular targeted therapies and immunotherapies, including small-molecule kinase inhibitors and monoclonal antibodies that target angiogenesis by inhibiting vascular endothelial growth factor (VEGF) signaling pathways. Despite their anti-angiogenic and anti-cancer benefits, the use of VEGF inhibitors (VEGFi) and other tyrosine kinase inhibitors (TKIs) has been hampered by potent vascular toxicities especially hypertension and thromboembolism. Molecular processes underlying VEGFi-induced vascular toxicities still remain unclear but inhibition of endothelial NO synthase (eNOS), reduced nitric oxide (NO) production, oxidative stress, activation of the endothelin system, and rarefaction have been implicated. However, the pathophysiological mechanisms still remain elusive and there is an urgent need to better understand exactly how anti-angiogenic drugs cause hypertension and other cardiovascular diseases (CVDs). This is especially important because VEGFi are increasingly being used in combination with other anti-cancer dugs, such as immunotherapies (immune checkpoint inhibitors (ICIs)), other TKIs, drugs that inhibit epigenetic processes (histone deacetylase (HDAC) inhibitor) and poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors, which may themselves induce cardiovascular injury. Here, we discuss vascular toxicities associated with TKIs, especially VEGFi, and provide an up-to-date overview on molecular mechanisms underlying VEGFi-induced vascular toxicity and cardiovascular sequelae. We also review the vascular effects of VEGFi when used in combination with other modern anti-cancer drugs.

Tissue sodium excess is not hypertonic and reflects extracellular volume expansion.

Our understanding of Na+ homeostasis has recently been reshaped by the notion of skin as a depot for Na+ accumulation in multiple cardiovascular diseases and risk factors. The proposed water-independent nature of tissue Na+ could induce local pathogenic changes, but lacks firm demonstration. Here, we show that tissue Na+ excess upon high Na+ intake is a systemic, rather than skin-specific, phenomenon reflecting architectural changes, i.e. a shift in the extracellular-to-intracellular compartments, due to a reduction of the intracellular or accumulation of water-paralleled Na+ in the extracellular space. We also demonstrate that this accumulation is unlikely to justify the observed development of experimental hypertension if it were water-independent. Finally, we show that this isotonic skin Na+ excess, reflecting subclinical oedema, occurs in hypertensive patients and in association with aging. The implications of our findings, questioning previous assumptions but also reinforcing the importance of tissue Na+ excess, are both mechanistic and clinical.

Epidermal growth factor signaling through transient receptor potential melastatin 7 cation channel regulates vascular smooth muscle cell function.

OBJECTIVE: Transient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function. Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR-TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice. CONCLUSIONS: We show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.

Oxidative Stress: A Unifying Paradigm in Hypertension.

The etiology of hypertension involves complex interactions among genetic, environmental, and pathophysiologic factors that influence many regulatory systems. Hypertension is characteristically associated with vascular dysfunction, cardiovascular remodelling, renal dysfunction, and stimulation of the sympathetic nervous system. Emerging evidence indicates that the immune system is also important and that activated immune cells migrate and accumulate in tissues promoting inflammation, fibrosis, and target-organ damage. Common to these processes is oxidative stress, defined as an imbalance between oxidants and antioxidants in favour of the oxidants that leads to a disruption of oxidation-reduction (redox) signalling and control and molecular damage. Physiologically, reactive oxygen species (ROS) act as signalling molecules and influence cell function through highly regulated redox-sensitive signal transduction. In hypertension, oxidative stress promotes posttranslational modification (oxidation and phosphorylation) of proteins and aberrant signalling with consequent cell and tissue damage. Many enzymatic systems generate ROS, but NADPH oxidases (Nox) are the major sources in cells of the heart, vessels, kidneys, and immune system. Expression and activity of Nox are increased in hypertension and are the major systems responsible for oxidative stress in cardiovascular disease. Here we provide a unifying concept where oxidative stress is a common mediator underlying pathophysiologic processes in hypertension. We focus on some novel concepts whereby ROS influence vascular function, aldosterone/mineralocorticoid actions, and immunoinflammation, all important processes contributing to the development of hypertension.

Acute vascular effects of vascular endothelial growth factor inhibition in the forearm arterial circulation.

OBJECTIVE: Although vascular endothelial growth factor inhibition (VEGFi) represents a major therapeutic advance in oncology, it is associated with hypertension and adverse vascular thrombotic events. Our objective was to determine whether VEGFi caused direct vascular dysfunction through increased endothelin-1 (ET-1) activity or impaired endothelial vasomotor or fibrinolytic function. METHODS: Using forearm venous occlusion plethysmography, we measured forearm blood flow during intra-arterial infusions of bevacizumab (36-144 µg/dl forearm volume per minute) administered for 15-60 min in healthy volunteers (n = 6-8). On two separate occasions in 10 healthy volunteers, we further measured forearm blood flow and tissue plasminogen activator (t-PA) release during intra-arterial bradykinin infusion (100 and 1000 pmol/min) in the presence and absence of bevacizumab (144 µg/dl forearm volume per minute), and the presence and absence of endothelin A receptor antagonism with BQ-123 (10 nmol/min). Plasma t-PA and plasminogen activator inhibitor-1 (PAI-1) concentrations were measured at baseline and with each dose of bradykinin. RESULTS: Baseline blood flow and plasma ET-1, t-PA and PAI-1 concentrations were unaffected by bevacizumab. Bradykinin caused dose-dependent vasodilatation (P < 0.0001) and t-PA release (P < 0.01) but had no effect on plasma PAI-1 concentrations. Neither bevacizumab nor BQ-123 affected bradykinin-induced vasodilatation and t-PA release. CONCLUSION: Acute exposure to bevacizumab does not directly cause endothelial vasomotor or fibrinolytic dysfunction in healthy young volunteers.

Selective ETA vs. dual ETA/B receptor blockade for the prevention of sunitinib-induced hypertension and albuminuria in WKY rats.

AIMS: Although effective in preventing tumour growth, angiogenesis inhibitors cause off-target effects including cardiovascular toxicity and renal injury, most likely via endothelin (ET)-1 up-regulation. ET-1 via stimulation of the ETA receptor has pro-hypertensive actions whereas stimulation of the ETB receptor can elicit both pro- or anti-hypertensive effects. In this study, our aim was to determine the efficacy of selective ETA vs. dual ETA/B receptor blockade for the prevention of angiogenesis inhibitor-induced hypertension and albuminuria. METHODS AND RESULTS: Male Wistar Kyoto (WKY) rats were treated with vehicle, sunitinib (angiogenesis inhibitor; 14 mg/kg/day) alone or in combination with macitentan (ETA/B receptor antagonist; 30 mg/kg/day) or sitaxentan (selective ETA receptor antagonist; 30 or 100 mg/kg/day) for 8 days. Compared with vehicle, sunitinib treatment caused a rapid and sustained increase in mean arterial pressure of ∼25 mmHg. Co-treatment with macitentan or sitaxentan abolished the pressor response to sunitinib. Sunitinib did not induce endothelial dysfunction. However, it was associated with increased aortic, mesenteric, and renal oxidative stress, an effect that was absent in mesenteric arteries of the macitentan and sitaxentan co-treated groups. Albuminuria was greater in the sunitinib- than vehicle-treated group. Co-treatment with sitaxentan, but not macitentan, prevented this increase in albuminuria. Sunitinib treatment increased circulating and urinary prostacyclin levels and had no effect on thromboxane levels. These increases in prostacyclin were blunted by co-treatment with sitaxentan. CONCLUSIONS: Our results demonstrate that both selective ETA and dual ETA/B receptor antagonism prevents sunitinib-induced hypertension, whereas sunitinib-induced albuminuria was only prevented by selective ETA receptor antagonism. In addition, our results uncover a role for prostacyclin in the development of these effects. In conclusion, selective ETA receptor antagonism is sufficient for the prevention of sunitinib-induced hypertension and renal injury.