Cardiovascular Research

Stroke and central nervous system dysfunction are cardinal symptoms in critically ill corona virus disease 19 (COVID-19) patients. In an autopsy series of 32 COVID-19 patients, we investigated whether carotid arteries were infected with SARS-CoV-2 by employing genomic, virologic, histochemical and transcriptomic analyses. We show that SARS-CoV-2 productively infects and modulates vascular responses in carotid arteries. This finding has far reaching implications for the understanding and clinical treatment of COVID-19.

Accumulation of oxalate in patients with chronic kidney disease (CKD) is associated with CKD progression and increased risk of cardiac death. Whether reducing plasma or urine oxalate slows CKD progression and prevents cardiovascular complications remains unexplored. We colonized the intestines of control and CKD mice with Oxalobacter formigenes (Oxf), an oxalate-degrading microorganism. The mice were fed with the oxalate precursor hydroxyproline for 23 weeks at which time we assessed pathological changes in the kidney and heart. We demonstrate that Oxf reduces plasma oxalate (pOx) and creatinine levels, mitigates inflammation and fibrosis in the kidney, and reduces pathologic cardiac remodeling in the hearts of CKD mice. RNA-seq analysis of ventricular tissue of CKD mice reveals dysregulated expression of metabolic pathways while Oxf colonization reverses these changes. These findings demonstrate that oxalate accumulation plays a role not only in CKD progression but also in associated cardiovascular complications and suggest that strategies to reduce plasma oxalate levels may have therapeutic benefit. Translational statementChronic kidney disease (CKD) is a major health problem that can lead to kidney failure and which increases the risk of cardiovascular disease (CVD) mortality. Oxalate accumulation in advanced kidney disease contributes to further CKD progression and CVD complications. Intestinal colonization with Oxalobacter formigenes (Oxf) in a CKD animal model reduces plasma oxalate level and slows progression of both CKD and CVD. Strategies to reduce plasma oxalate levels may have therapeutic benefit in the setting of CKD.

IntroThe prevalence of abdominal aortic aneurysm (AAA) is constantly progressing with the aging of the global population. AAA rupture has a devastating 80% mortality rate and there is no treatment to slow-down AAA progression. Hydrogen sulfide (H2S) is a ubiquitous redox-modifying gasotransmitter produced in the cardiovascular system via the reverse trans-sulfuration pathway by cystathionine {gamma}-lyase (CSE). H2S has protective properties on the cardiovascular system, including anti-inflammatory and antioxidant effects. Here, we hypothesized that sodium thiosulfate (STS), a clinically relevant source of H2S, would limit AAA growth. Methods8-12 weeks old male WT or Cse-/- mice on a C57BL/6J genetic background were submitted to a model of AAA by topical elastase application on the abdominal aorta and {beta}-aminopropionitrile fumarate treatment in the drinking water for 2 weeks post-op. Sodium thiosulfate (STS) was given via the drinking water post-op until aorta collection. In vitro experiments were conducted to assess the effect of STS and pro-inflammatory cytokines interleukin-1 {beta} and 6 and tumor necrosis factor on primary human vascular smooth muscle cell (VSMC). ResultsSurprisingly, STS increased elastin degradation, AAA size and rupture, despite reducing infiltration of macrophages, antigen-presenting cells and lymphocytes in WT mice. Conversely, Cse-/- mice with impaired H2S production developed smaller AAA than WT mice despite increased infiltration of immune cells. STS reduced VSMC coverage, possibly lowered VSMC proliferation, and promoted VSMC loss and extracellular matrix (ECM) breakdown. In vitro, STS aggravated pro-inflammatory cytokine-induced VSMCs apoptosis. ConclusionSTS has a paradoxical effect on AAA growth, reducing inflammation while simultaneously impeding favorable vascular remodeling, resulting in bigger AAA in a model of periadventitial elastase. This study identifies a negative effect of H2S on VSMC in this environment, highlighting the complex role of H2S in AAA progression. The deleterious effect of STS on AAA progression is significant, especially given the growing use of STS in clinical settings for various indications.

Backgroundglobal deletion of Vhl leads to vascular defects and early lethality, precluding the study of VHL/HIF signaling during coronary formation and homeostasis. Hypoxia pathway has been associated with cardiovascular diseases involving inflammation and vascular remodeling like atherosclerosis, but its role in Kawasaki Disease (KD) remains unknown. Coronary dilatation and vessel rupture are the most serious complications of KD, while the molecular mechanisms underlying these cardiac events remain poorly understood. Here we aim to determine the function of VHL/HIF pathway in the development of cardiovascular defects and its role in KD. MethodsWe generated a new mouse model to genetically hyperactivate hypoxia pathway in progenitors contributing to coronary vessels and cardiac fibroblasts (Vhl/Wt1). We characterized the model by means of echocardiography, magnetic resonance imaging, histological analysis and molecular approaches. Human cardiac tissue from KD individuals suffering fatal coronary aneurysm were screened for HIF signaling and inflammatory markers by immunohistochemistry. Resultsconditional Vhl KO do not undergo developmental abnormalities but displays cardiomegaly and epicardial vascular defects, with cardiac hypertrophy and progressive coronary diameter increase, as well as pericardial hemorrhage and systemic inflammation early after birth. Histological characterization reveals inflammation of coronary arteries, vascular remodeling with elastin breaks and dilatation, increased perivascular fibrosis and smooth muscle cells death, together with high incidence of intracoronary thrombus formation. In addition, the mutants display vascular calcification and severe cardiac inflammation and interstitial hemorrhages, dying suddenly between 15-20 weeks of age due to vessel rupture. Simultaneous elimination of HIF2 and VHL prevents the cardiovascular abnormalities displayed by single cVhl KO, highlighting the essential role of HIF2 in coronary instability and vascular inflammation. Histological characterization of human cardiac samples shows positive signal for HIF1 and specially HIF2, in the coronary lesions and its surroundings in regions with high inflammatory infiltration, confirming the activation of hypoxia signaling in KD patients with cardiovascular complications. ConclusionsOur data demonstrate the importance of HIF2 signaling in the development of coronary inflammation and vascular remodeling and provide new evidences connecting low oxygen tensions with cardiovascular lesions occurring during the onset of the most severe cases of KD. Furthermore, the Vhl/Wt1 mouse generated recapitulates cardiac features of KD with critical heart complications, providing a new platform to uncover unknown aspects of KD pathogenesis.

Unstructured AbstractPrimary aldosteronism (PA) is renin-independent aldosterone production that causes hypertension and cardiovascular disease. We investigated the proteomic evolution of PA from normotensive people with renin-independent aldosteronism to those with overt PA. The PA plasma proteome was characterized by pathways related to cardiovascular disease (inflammation, energy/redox, vascular remodeling). We identified proteins exhibiting dose-dependent trends paralleling the continuum of renin-independent aldosterone production, then using adrenal vein proteomics, identified proteins exhibiting the archetypal pattern of unilateral PA (peak abundance in the dominant vein with suppression in the contralateral vein). Among these, Norrin, a Wnt/{beta}-catenin ligand previously identified as a risk locus for PA by GWAS, was robustly validated using functional testing (ACTH- and angiotensin II-induced interventions, and correlations with 18-hybrid steroids) and genetic testing (dose-dependent associations with NDP SNPs). The evolution of PA originates in normotensive people, is characterized by proteomic signatures of cardiovascular disease, and Norrin is a novel regulator of PA pathophysiology.

Although respiratory symptoms are the most prevalent disease manifestation of infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), nearly 20% of hospitalized patients are at risk for thromboembolic events. This prothrombotic state is considered a key factor in the increased risk of stroke, which is observed clinically during both acute infection and long after symptoms clear. Here we develop a model of SARS-CoV-2 infection using human-induced pluripotent stem cell-derived endothelial cells (ECs), pericytes (PCs), and smooth muscle cells (SMCs) to recapitulate the vascular pathology associated with SARS-CoV-2 exposure. Our results demonstrate that perivascular cells, particularly SMCs, are a susceptible vascular target for SARS-CoV-2 infection. Utilizing RNA sequencing, we characterize the transcriptomic changes accompanying SARS-CoV-2 infection of SMCs, PCs, and ECs. We observe that infected SMCs shift to a pro-inflammatory state and increase the expression of key mediators of the coagulation cascade. Further, we show human ECs exposed to the secretome of infected SMCs produce hemostatic factors that contribute to vascular dysfunction, despite not being susceptible to direct infection. The findings here recapitulate observations from patient sera in human COVID-19 patients and provide mechanistic insight into the unique vascular implications of SARS-CoV-2 infection at a cellular level.

BackgroundHypertension is a leading cause of microvascular injury, yet the genetic determinants of organ-specific vulnerability remain poorly understood. Yest, we need good mouse models to investigate the complication of hypertension. This study investigates the role of genetic background in shaping hypertensive complications by comparing two mouse strains with divergent inflammatory responses. MethodsThree-month-old 129S1/SvlmJ and C57BL/6J mice received 600 ng/kg/min of angiotensin II (AngII) or saline. We compared the consequences of ANG2-induced blood pressure elevation on kidney function, BBB intergrity and cardiac hypertropy. Blood pressure (BP) was assessed by telemetry. Vascular injury markers in the brain, heart, kidneys, and retinas were systematically evaluated. ResultsBoth strains developed similar moderate hypertension with AngII. Only 129S1/SvlmJ mice exhibited spatial learning and memory deficits, blood-brain barrier hyperpermeability, astrocyte activation, retinal artery damage, hypertrophic cardiomyopathy, and renal podocyte lesions with urinary albumin/creatinine ratio (UACR) after AngII treatment. Transcriptomic analysis of brain microvessels highlighted strain-specific differences in gene regulation, particularly in inflammatory pathways, which may explain the higher vulnerability of 129S1/SvlmJ mice to hypertensive organ damage. These findings were supported in vivo by increased resident and perivascular macrophage recruitment in the brain of C57BL6/J mice under AngII compared to the 129S1/SvlmJ strain. ConclusionOur findings highlight the critical role of genetic background in shaping hypertensive complications. The 129S1/SvlmJ strain serves as a valuable model for dissecting the molecular mechanisms of hypertensive organ damage, emphasizing neurovascular inflammation as a potential therapeutic target. Translational PerspectiveThis study highlights the 129/Sv mouse strain as a superior translational model compared to the widely used C57BL/6J strain, which, despite being a standard in cardiovascular research, fails to reliably reproduce severe hypertensive organ complications. The 129/Sv strain closely mimics human hypertensive damage, including cerebral small vessel disease, nephropathy, cardiomyopathy, and retinopathy. Transcriptomic analysis of cerebral microvessels identifies maladaptive inflammation as a critical mechanistic driver of susceptibility. These findings underline the clinical relevance of genetic predisposition, improving risk stratification and providing a robust preclinical platform to develop targeted anti-inflammatory therapies aimed at preventing hypertension-induced end-organ damage in patients.

Accumulating clinical observations implicate vascular inflammation as an underlying cause of coagulopathy in severely ill COVID-19 patients and it was recently suggested that SARS-CoV-2 virus particles infect endothelial cells. Here, we show that endothelial cells do not express angiotensin-converting enzyme-2 (ACE2), the SARS-CoV-2 receptor. Instead, pericytes and microvascular smooth muscle cells express ACE2 in an organotypic manner. Pericyte deficiency leads to increased endothelial expression and release of Von Willebrand factor and intravascular platelet and fibrin aggregation, suggesting that pericytes limit endothelial pro-thrombotic responses. That pericytes and not endothelial cells express ACE2 may provide important clues to the pathology of COVID-19, as pericytes are normally shielded behind an endothelial barrier and may get infected only when this barrier is compromised by COVID-19 risk factors.

The vascular barrier is heavily injured following SARS-CoV-2 infection and contributes enormously to life-threatening complications in COVID-19. This endothelial dysfunction is associated with the phlogistic phenomenon of cytokine storms, thrombotic complications, abnormal coagulation, hypoxemia, and multiple organ failure. The mechanisms surrounding COVID-19 associated endotheliitis have been widely attributed to ACE2-mediated pathways. However, integrins have emerged as possible receptor candidates for SARS-CoV-2, and their complex intracellular signalling events are essential for maintaining endothelial homeostasis. Here, we showed that the spike protein of SARS-CoV-2 depends on its RGD motif to drive barrier dysregulation through hijacking integrin V{beta}3. This triggers the redistribution and internalization of major junction protein VE-Cadherin which leads to the barrier disruption phenotype. Both extracellular and intracellular inhibitors of integrin V{beta}3 prevented these effects, similarly to the RGD-cyclic peptide compound Cilengitide, which suggests that the spike protein - through its RGD motif - binds to V{beta}3 and elicits vascular leakage events. These findings support integrins as an additional receptor for SARS-CoV-2, particularly as integrin engagement can elucidate many of the adverse endothelial dysfunction events that stem from COVID-19.

Acute mesenteric ischemia (AMI) remains associated with high mortality despite prompt revascularization, suggesting that downstream ischemia-reperfusion injury contributes to poor outcomes. However, the microvascular mechanisms underlying this process remain poorly defined. We analyzed admission blood samples from patients with arterial AMI and non-ischemic controls and investigated thrombo-inflammatory responses in a murine superior mesenteric artery occlusion (SMAO) model. In mice, intravital microscopy was used to directly visualize mesenteric microcirculatory flow and thrombo-inflammatory events during ischemia-reperfusion. Patients with AMI displayed a marked systemic thrombo-inflammatory profile, characterized by elevated inflammatory markers, neutrophil activation, platelet activation, and alterations in coagulation-related proteins, which were closely mirrored in the SMAO model. Intravital microscopy revealed a dissociation between arterial and microvascular reperfusion: while arteriolar flow partially recovered after recanalization, venular perfusion remained severely impaired and was associated with early blood cell stasis and stable venular thrombus formation. Thrombi developed through a sequential process initiated during ischemia and amplified during reperfusion. Together, these findings identify venular-centered thrombo-inflammation as a key determinant of microvascular dysfunction and intestinal injury in arterial AMI, and provide a mechanistic framework for targeting thrombo-inflammatory pathways beyond arterial reperfusion that may extend to other clinical forms of AMI, including non-occlusive mesenteric ischemia (NOMI).

BackgroundHypertensive emergency (HTEM) is defined by abrupt blood pressure elevation with acute multi-organ damage, yet the mechanisms predisposing only a subset of hypertensive individuals to HTEM remain unclear. Progress has been limited by the lack of a mouse model that faithfully replicates human disease. We aimed to identify determinants of susceptibility to hypertensive microvascular injury and characterize a murine model of HTEM. MethodsMale C57BL/6J (B6J) and 129S2/SvPasCrl (129Sv) mice were exposed to severe hypertension via angiotensin II infusion combined with a high-salt diet. We assessed survival, renal and retinal injury, cardiac function and electrophysiology, vascular permeability, circulating angiogenic factors, and glomerular transcriptional profiles using single-cell RNA sequencing. Bone marrow transplantation and recombinant human PlGF-2 treatment were used to investigate mechanisms driving endothelial injury. ResultsDespite comparable blood pressure, 129Sv mice, but not B6J, developed malignant hypertension with albuminuria, acute kidney injury, retinal hemorrhages, microvascular leakage, cardiac dysfunction, and arrhythmias. Hypertensive 129Sv mice exhibited markedly elevated circulating sFlt-1. PlGF-2 supplementation partially reversed albuminuria, preserved glomerular ultrastructure, and reduced retinal hemorrhages. Bone marrow transfers revealed contributions from both hematopoietic and non-hematopoietic 129Sv compartments to sFlt-1 overproduction and organ injury. Single-cell transcriptomics revealed profound repression of angiogenic, metabolic, and stress-response pathways in glomerular endothelial cells, a repression partially restored by PlGF-2. ConclusionsWe identify 129Sv mice as a robust model of HTEM, exhibiting multi-organ microvascular injury that closely mirrors the human condition. Our results reveal blood-pressure-independent susceptibility to organ damage and implicate dysregulated VEGFA/sFlt-1 signaling as a central driver of endothelial dysfunction, highlighting angiogenic imbalance as a potential therapeutic target.

In the last years, autophagy has emerged as an essential pathway for most cellular functions. Basal autophagy plays a protective role as a quality control mechanism by which damaged or noxious cellular components are degraded and cellular organelles are periodically renewed. Moreover, autophagic activity can be increased in situations of cellular stress, including nutrient or growth factor deprivation, hypoxia, reactive oxygen species, DNA damage, or the presence of intracellular pathogens. Normally, induction of autophagy is protective, although in some circumstances, such as conditions of hemodynamic stress, autophagosome accumulation upon autophagy induction can be a maladaptive process. The deficiency of the autophagic protease ATG4D in mice leads to the accumulation of cellular autophagosomes in most tissues, including the heart. Here, we show that the increased autophagosome content of atg4d-/- mice is linked to the development of late-onset cardiomyopathy and to increased susceptibility to heart failure induced by transverse aortic constriction. Furthermore, we report the existence of human ATG4D variants associated with cardiovascular pathologies and also that ATG4D expression is reduced in human obstructive hypertrophic cardiomyopathy and dilated cardiomyopathy, which highlights a conserved cardio-protective role of the ATG4D protease.

BackgroundThoracic aortic aneurysm (TAA) is a life-threatening condition with an unpredictable lisk of rupture. Current clinical parameters have limited ability to accurately predict imminent rupture. Osteopontin (OPN) has been implicated in aortic aneurysm pathology, however, it role as a marker of imminent rupture remains. unclear. We investigated the dynamics of OPN expression dynamics in a mouse model with predictable rupture timing and validated our findings in human TAA. MethodsOne-month-old fibrillin-1 hypomorphic (Fbn1mgR/mgR) mice were used as a TAA model; with wild-type (WT) mice served as controls. Angiotensin II (AngII) was administered to Fbn1mgR/mgR to induce acute aortic rupture. Single-section transcriptome analysis and immunofluorescence staining were performed on ascending aortic tissue at 24 and 72 hours after AngII infusion, with pre-treatment Fbn1mgR/mgR and WT mice serving as controls. To determine conservation in human disease, we reanalyzed publicly available single-cell RNA sequencing data from ascending thoracic aortic aneurysm (ATAA) patients. ResultsAngII infusion induced progressive mortality beginning at 24 hours, with approximately 60% survival at 72 hours and nearly no survival by 8 days in Fbn1mgR/mgR mice. At this pre-rupture time point, OPN showed prominent upregulation at both mRNA and protein levels in ascending aortic tissues compared to controls. Immunofluorescence staining revealed increased OPN expression in the aortic wall, particularly in regions exhibiting structural deterioration. Reanalysis of human ATAA single-cell data showed elevated OPN expression compared to controls, with enrichment in immune cell populations, especially macrophages. Within the macrophage compartment, subcluster analysis identified a stress-responsive subpopulation (MC1) that was markedly expanded and almost exclusively composed of ATAA-derived cells, representing the primary source of OPN upregulation. ConclusionsOPN upregulation represents a conserved molecular signature of the pre-rupture state in TAA across mice and humans. Our mode, which enables predictable rupture timing, allowed the capture of acute pre-rupture molecular changes, suggesting OPN as a potential biomarker for predicting imminent aortic rupture.

Glycosaminoglycans (GAGs) pooling has been considered since long as one of the histopathological characteristics defining thoracic aortic aneurysm (TAA) together with smooth muscle cells (SMCs) apoptosis and elastin fibers degradation. However, few information is provided about GAGs composition or potential implication in TAA pathology. Syndecan-1 (Sdc-1) is a heparan sulfate proteoglycan that is implicated in extracellular matrix (ECM) interaction and assembly, regulation of SMCs phenotype and various aspects of inflammation in the vascular wall. In the current work, the regulation of Sdc-1 protein was examined in human TAA by ELISA and immunohistochemistry. In addition, the role of Sdc-1 was evaluated in descending TAA in vivo using a mouse model combining both aortic wall weakening and hypertension. Our results showed that Sdc-1 protein is over expressed in human TAA aortas compared to healthy counterparts and that SMCs are the major cell type expressing Sdc-1. Similarly, in the mouse model used, Sdc-1 expression was increased in TAA aortas compared to healthy samples. Although its protective role against abdominal aneurysm has been reported, we observed that Sdc-1 was dispensable for TAA prevalence or rupture. In addition, Sdc-1 deficiency did not alter the extent of aortic wall dilatation, elastin degradation, collagen deposition, or leukocyte recruitment in our TAA model. These findings suggest that Sdc-1 could be a biomarker revealing TAA pathology. Future investigations could uncover the underlying mechanisms leading to Sdc-1 expression alteration in TAA.

RationaleSuccessful and timely coronary reperfusion following acute ST-elevation myocardial infarction (STEMI) is standard therapy to salvage transiently ischaemic heart muscle. However, the subsequent inflammatory response within the infarct can lead to further loss of viable myocardium. Robust interventions are required in the acute MI setting to minimise cardiac injury and reduce risk of further detrimental progression. ObjectiveTGF{beta}1 is an anti-inflammatory cytokine released endogenously in response to infection or tissue injury. The goal of this study was to investigate its protective effects when given exogenously following myocardial infarction. Methods and ResultsTGF{beta}1 is found at increased levels in the blood of STEMI patients immediately following myocardial infarction. We observe a significant correlation (p=0.003) between higher circulating TGF{beta}1 levels at 24h post MI and a reduction in infarct size over the following 3 months, suggesting that an early increase in circulating TGF{beta}1 is protective in these patients. Using a mouse model of cardiac ischaemia-reperfusion we demonstrate that additional exogenous TGF{beta}1 delivered in the acute setting has multiple beneficial outcomes. At 24 hours post-reperfusion It leads to a significantly smaller infarct size (30% reduction, p=0.025), reduced inflammatory infiltrate (28% reduction, p=0.015), lower intra-cardiac expression of inflammatory cytokines IL1{beta} and CCL2 (>50 % reduction, p=0.038 and 0.0004, respectively) and reduced scar size at 4 weeks (21% reduction, p=0.015). Furthermore exogenous delivery of an equivalent dose of HpTGM, a recently described low-fibrogenic mimic of TGF{beta}1, secreted by a helminth parasite to evade immune rejection, has an almost identical protective effect on injured mouse hearts. Furthermore using a genetic approach we show the benefit is mediated by the vascular endothelium. ConclusionsThis work reveals the potential of exogenous TGF{beta}1 and HpTGM delivered in the acute MI setting to provide protective anti-inflammatory effects and reduce infarct size, leading to a smaller scar and reduced detrimental progression.

BackgroundAortic aneurysm and dissection (AAD) are highly lethal conditions for which hypertension serves as a primary risk factor. The limited efficacy of conventional antihypertensive treatments suggests an inadequate understanding of the molecular mechanisms that connect these conditions. Despite their pivotal role in regulating biological functions, the specific proteomic signatures associated with both hypertension and AAD have not been extensively investigated. This study sought to conduct a comprehensive mapping of the plasma proteome to identify novel biomarkers and therapeutic targets for AAD within a hypertensive cohort. MethodsWe analyzed 2,923 plasma proteins in 26,690 hypertensive individuals without a prior history of AAD from the UK Biobank. LASSO and Cox regression analyses were employed to identify proteins associated with AAD, while a LightGBM algorithm was utilized to construct predictive models. Causal inference was conducted using two-sample Mendelian randomization (MR). Further mechanistic exploration included colocalization, single-cell RNA sequencing, functional enrichment, and drug-target analysis. ResultsAmong the core hypertension proteins, we identified 186 proteins independently associated with AAD risk, with MMP12 showing the strongest association. A streamlined model, which included the top five proteins alongside age and sex, exhibited superior predictive performance (AUC: 0.791) compared to traditional risk models. MR analysis confirmed causal relationships for 21 proteins with AAD, and colocalization provided high-confidence evidence for shared genetic architecture for MMP7, CCN3, and COL6A3. Mechanistically, single-cell analysis verified cell-type-specific aortic expression of candidate genes, functional enrichment implicated extracellular matrix (ECM) pathways. Furthermore, we identified 95 FDA-approved drugs targeting 11 of these causal proteins. ConclusionThis study presents the first comprehensive plasma proteomic landscape of AAD within a large hypertensive cohort, offering a high-performance predictive model, validating novel causal proteins, and identifying actionable drug targets. These findings provide crucial molecular insights into the pathogenesis of AAD and establish a solid foundation for developing early-detection strategies, improving risk stratification, and guiding precision medicine.

BackgroundWhile roles for canonical autophagy in the pathophysiology of cardiovascular disease have been established, we have limited understanding of the non-canonical functions of autophagy proteins in this context. LC3-asssociated endocytosis (LANDO) is a novel non-canonical function of autophagy proteins, in which LC3 (microtubule-associated protein light chain 3) is conjugated to early endosome membranes using a portion of the canonical autophagy machinery, and functions in the endocytic recycling of several plasma membrane proteins. Here we ask whether perturbation of LANDO can promote cardiovascular pathogenesis. MethodsCardiac and endothelial functions were assessed by echocardiography and flow-mediated dilatation in mice lacking Rubicon (Rubcn-/-) or the WD domain of ATG16L1 (Atg16l1{Delta}WDki), two known effectors of LANDO. Mice with conditional depletion of Rubicon in the endothelial, myeloid and cardiomyocyte compartments were used as well. Three-dimensional murine cardiac vasculature leakiness was investigated by light sheet fluorescence microcopy. Endothelial activation induced by shear stress was characterized in vitro in primary endothelial cells isolated from murine lungs and human aortic endothelial cells. Associations between genetically predicted expression of candidate genes involved in LANDO and human cardiovascular parameters were studied in the Young Finns Study and the UK Biobank. ResultsCompared to littermate controls, young Rubcn-/- and Atg16l1{Delta}WDki mice showed a decrease in cardiac and endothelial functions, as did mice with endothelium-specific deficiency. VEGFR2 recycling to the plasma membrane and nitric oxide pathway during shear stress were disrupted in LANDO-deficient primary murine and human endothelial cells. Proteomic analysis in primary human aortic endothelial cells revealed an upregulation of intracellular hemoglobin subunit alpha (Hb-) upon shear stress, which was blunted when RUBCN was ablated. Genetic expression studies uncovered several candidate genes related to LANDO that correlated with cardiovascular parameters. These included the retromer complex subunit VPS29, disruption of which decreased Hb- expression levels in human endothelial cells. ConclusionsOur data support a pivotal role of non-canonical functions of autophagy proteins in recycling VEGFR2 upon shear stress activation in endothelial cells together with Hb- expression that may contribute to the etiology of cardiovascular diseases.

Failure to reperfuse the coronary microvasculature ("no-reflow") affects up to 50% of patients after unblocking a coronary artery that caused ischaemia and acute myocardial infarction. No-reflow is associated with reduced left ventricular ejection fraction, increased infarct size and death. We have established that no-reflow results from cardiac pericytes constricting coronary capillaries, and that pharmacologically relaxing pericytes reduces no-reflow. Remote ischaemic preconditioning, by briefly making a limb ischaemic, protects against cardiac ischaemic injury, and we have shown this is mediated by release of the gut hormone glucagon-like peptide 1 (GLP-1). We now demonstrate that, by releasing GLP-1, remote ischaemic preconditioning reduces pericyte-mediated coronary capillary constriction and no-reflow, and that the dilating effect of GLP-1 on coronary capillaries is abolished by block or genetic deletion of pericyte KATP channels. These results define a brain-gut-heart pathway mediating remote ischaemic cardioprotection, and suggest pharmacological therapies to reduce ischaemia-induced coronary no-reflow and improve post-infarct recovery.

BackgroundCarotid intima-media thickening (IMT) is a major risk factor for cardiovascular disease (CVD). The large ribosomal subunit protein 17 (Rpl17) was recently reported as a CVD-associated gene; however, ribosomal mutations generally are not associated with vascular dysfunction. We have created a novel genetic model of decreased RpL17 in endothelial cells (EC) to determine how changes in endothelial ribosome expression cause IMT. MethodsEC-restricted RpL17 heterozygous mice (Cdh5-Cre; RpL17fl/wt, or Rpl17-Het), were generated and subjected to sham or partial carotid ligation (PCL) surgery of the left artery to induce acute disturbed (d)-flow in vivo. Carotids were harvested on day 14 for quantitative tissue immunostaining. Purified mouse and human EC in vitro were exposed to steady (s)-flow or d-flow using cone viscometry, and collected for flow cytometry, protein expression, electron microscopy, or purification of ribosomes. Human carotid samples from healthy and endarterectomy patients were used for tissue analysis. ResultsCarotids from RpL17-Het mice with PCL-induced d-flow showed increased IMT relative to RpL17-WT controls. In addition, RpL17 protein levels were decreased in regions of d-flow compared to s-flow. Increased levels of ER stress markers were observed by carotid immunostaining, as well as activation of the integrated stress response (ISR) in RpL17-Het EC. Analysis of mRNAs bound to polysomes vs. monosomes in EC-RpL17-Het revealed increased translational efficiency of key regulators of glycolysis, redox, inflammation, matrix, and endothelial-to-mesenchymal transition (EndMT). Metabolic profiling by Seahorse assay showed enhanced anaerobic glycolysis and decreased oxidative respiration in RpL17-Het EC, consistent with the translational efficiency data. Immunostaining of carotids identified upregulated EC inflammation and EndMT. ConclusionsOur data support RpL17 as a key mediator of EC phenotypic modulation that causes IMT in response to d-flow. We show a novel pathway for d-flow-mediated IMT: endoplasmic reticulum stress and activation of the ISR. These changes alter translational efficiency and reprogram EC cell cycle, metabolism, and redox state in the presence of d-flow to cause IMT, a precursor to cardiovascular pathology.

We address a paradox, that AMPK may facilitate hypoxic pulmonary vasoconstriction and its deficiency precipitate pulmonary hypertension. Here we show that AMPK-1/2 deficiency in smooth muscles promotes persistent pulmonary hypertension of the newborn. Accordingly, dual AMPK-1/2 deletion in smooth muscles causes premature death of mice after birth, associated with increased muscularization and remodeling throughout the pulmonary arterial tree, reduced alveolar numbers and alveolar membrane thickening, but with no edema. Spectral Doppler ultrasound indicates pulmonary hypertension and attenuated hypoxic pulmonary vasoconstriction. Age-dependent right ventricular pressure elevation, dilation and reduced cardiac output was also evident. KV1.5 potassium currents of pulmonary arterial myocytes are markedly smaller under normoxia, which is known to facilitate pulmonary hypertension. Mitochondrial fragmentation and reactive oxygen species accumulation is also evident. Importantly, there is no evidence of systemic vasculopathy or hypertension in these mice. Moreover, hypoxic pulmonary vasoconstriction is attenuated by AMPK-1 or AMPK-2 deletion without triggering pulmonary hypertension.