NAD+ exhaustion by CD38 upregulation contributes to blood pressure elevation and vascular damage in hypertension.

Hypertension is characterized by endothelial dysfunction and arterial stiffness, which contribute to the pathogenesis of atherosclerotic cardiovascular diseases. Nicotinamide adenine dinucleotide (NAD+) is an indispensable cofactor in all living cells that is involved in fundamental biological processes. However, in hypertensive patients, alterations in NAD+ levels and their relation with blood pressure (BP) elevation and vascular damage have not yet been studied. Here we reported that hypertensive patients exhibited lower NAD+ levels, as detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS), in both peripheral blood mononuclear cells (PBMCs) and aortas, which was parallel to vascular dysfunction. NAD+ boosting therapy with nicotinamide mononucleotide (NMN) supplement reduced BP and ameliorated vascular dysfunction in hypertensive patients (NCT04903210) and AngII-induced hypertensive mice. Upregulation of CD38 in endothelial cells led to endothelial NAD+ exhaustion by reducing NMN bioavailability. Pro-inflammatory macrophages infiltration and increase in IL-1ß generation derived from pro-inflammatory macrophages resulted in higher CD38 expression by activating JAK1-STAT1 signaling pathway. CD38 KO, CD38 inhibitors treatment, or adeno-associated virus (AAV)-mediated endothelial CD38 knockdown lowered BP and improved vascular dysfunction in AngII-induced hypertensive mice. The present study demonstrated for the first time that endothelial CD38 activation and subsequently accelerated NAD+ degradation due to enhanced macrophage-derived IL-1ß production was responsible for BP elevation and vascular damage in hypertension. NAD+ boosting therapy can be used as a novel therapeutic strategy for the management of hypertensive patients.

Neck-to-height ratio and arterial stiffness in Chinese adults: cross-sectional associations in a community-based cohort.

OBJECTIVES: The aim of this study was to investigate the association between neck-to-height ratio (NHR) and arterial stiffness in adults from a community-based Chinese cohort in a cross-sectional study. METHODS: We conducted cross-sectional analysis using data from the Kailuan study, a population-based cohort research. Altogether, 18 972 individuals were included in the analysis. Brachial ankle pulse wave velocity (baPWV), anthropometric indexes and cardiovascular risk factors were recorded. Data were analyzed by multiple lineal regression model. RESULTS: NHR was positively associated with baPWV after adjusted for age, sex, blood pressure, heart rate, BMI, waist-hip ratio, current smoking, fasting blood glucose, serum cholesterol, uric acid, high-sensitivity C reactive protein and creatinine clearance (ß = 5.76, P < 0.001), while the association of neck circumference and baPWV was NS after adjusting the variables mentioned above. In subgroups analysis, the association between NHR and baPWV did not reach statistical significance in female, while in males, the association was significant. Interaction effects were observed among BMI stratifications and the individuals with metabolic syndrome and history of cardiovascular events (P for intereaction = 0.002, 0.038 and 0.003, respectively). CONCLUSION: The current study demonstrated for the first time that NHR was positively associated with baPWV in community-based population, NHR might be a promising independent predictor for cardiovascular disease.

Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart.

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRSL274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Methods and Results: Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton. Conclusions: MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

Sam68 impedes the recovery of arterial injury by augmenting inflammatory response.

OBJECTIVE: The role of Src-associated-in-mitosis-68-kDa (Sam68) in cardiovascular biology has not been studied. A recent report suggests that Sam68 promotes TNF-α-induced NF-κB activation in fibroblasts. Here we sought to dissect the molecular mechanism by which Sam68 regulates NF-κB signaling and its functional significance in vascular injury. APPROACH AND RESULTS: The endothelial denudation injury was induced in the carotid artery of Sam68-null (Sam68-/-) and WT mice. Sam68-/- mice displayed an accelerated re-endothelialization and attenuated neointima hyperplasia, which was associated with a reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines in the injured vessels. Remarkably, the ameliorated vascular remodeling was recapitulated in WT mice after receiving transplantation of bone marrow (BM) from Sam68-/- mice, suggesting the effect was attributable to BM-derived inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-α-induced expression of TNF-α, IL-1ß, and IL-6 and in the level of nuclear phospho-p65, indicating attenuated NF-κB activation; and these results were confirmed in peritoneal and BM-derived macrophages of Sam68-/- vs. WT mice. Furthermore, co-immunoprecipitation and mass-spectrometry identified Filamin A (FLNA) as a novel Sam68-interacting protein upon TNF-α treatment. Loss- and gain-of-function experiments suggest that Sam68 and FLNA are mutually dependent for NF-κB activation and pro-inflammatory cytokine expression, and that the N-terminus of Sam68 is required for TRAF2-FLNA interaction. CONCLUSIONS: Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery by interacting with FLNA to stabilize TRAF2 on the cytoskeleton and consequently potentiate NF-κB signaling.

E2F1 Suppresses Oxidative Metabolism and Endothelial Differentiation of Bone Marrow Progenitor Cells.

RATIONALE: The majority of current cardiovascular cell therapy trials use bone marrow progenitor cells (BM PCs) and achieve only modest efficacy; the limited potential of these cells to differentiate into endothelial-lineage cells is one of the major barriers to the success of this promising therapy. We have previously reported that the E2F transcription factor 1 (E2F1) is a repressor of revascularization after ischemic injury. OBJECTIVE: We sought to define the role of E2F1 in the regulation of BM PC function. METHODS AND RESULTS: Ablation of E2F1 (E2F1 deficient) in mouse BM PCs increases oxidative metabolism and reduces lactate production, resulting in enhanced endothelial differentiation. The metabolic switch in E2F1-deficient BM PCs is mediated by a reduction in the expression of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase kinase 2; overexpression of pyruvate dehydrogenase kinase 4 reverses the enhancement of oxidative metabolism and endothelial differentiation. Deletion of E2F1 in the BM increases the amount of PC-derived endothelial cells in the ischemic myocardium, enhances vascular growth, reduces infarct size, and improves cardiac function after myocardial infarction. CONCLUSION: Our results suggest a novel mechanism by which E2F1 mediates the metabolic control of BM PC differentiation, and strategies that inhibit E2F1 or enhance oxidative metabolism in BM PCs may improve the effectiveness of cell therapy.

CXCR7 upregulation is required for early endothelial progenitor cell-mediated endothelial repair in patients with hypertension.

Dysfunction of early endothelial progenitor cells (EPCs) is responsible for impaired endothelial repair capacity after arterial injury in patients with hypertension. Here, we hypothesized that diminished signaling of CXC chemokine receptor 7 (CXCR7) contributes to the reduced EPC functions, and enhanced CXCR7 expression restores the capacities of EPCs from hypertensive patients. CXCR7 expression of EPCs from hypertensive patients was significantly reduced when compared with that from healthy subjects. Meanwhile, the phosphorylation of p38 mitogen-activated protein kinase, a downstream signaling of CXCR7, was elevated, which increased cleaved caspase-3 level of EPCs. CXCR7 gene transfer augmented CXCR7 expression and decreased the phosphorylation of p38 mitogen-activated protein kinase, which was paralleled to EPC functional upregulation of in vitro adhesion, antiapoptosis activities, and in vivo re-endothelialization capacity in a nude mouse model of carotid artery injury. The enhanced in vitro and in vivo functions of EPCs were markedly inhibited by neutralizing monoclonal antibody against CXCR7, which was blocked by p38 mitogen-activated protein kinase inhibitor SB203580. Downregulation of cleaved caspase-3 level induced by CXCR7 gene transfer or SB203580 pretreatment improved EPC functions. Furthermore, we found that lercanidipine, a dihydropyridine calcium channel antagonist, enhanced CXCR7 expression and facilitated in vitro and in vivo functions of EPCs. Our study demonstrated for the first time that diminished CXCR7 signal at least partially contributes to the reduced in vitro functions and in vivo re-endothelialization capacity of EPCs from hypertensive patients. Upregulation of CXCR7 expression induced by gene transfer or lercanidipine treatment may be a novel therapeutic target for increased endothelial repair capacity in hypertension.

Contrasting roles of E2F2 and E2F3 in cardiac neovascularization.

Insufficient neovascularization, characterized by poor endothelial cell (EC) growth, contributes to the pathogenesis of ischemic heart disease and limits cardiac tissue preservation and regeneration. The E2F family of transcription factors are critical regulators of the genes responsible for cell-cycle progression and growth; however, the specific roles of individual E2Fs in ECs are not well understood. Here we investigated the roles of E2F2 and E2F3 in EC growth, angiogenesis, and their functional impact on myocardial infarction (MI). An endothelial-specific E2F3-deficient mouse strain VE-Cre; E2F3(fl/fl) was generated, and MI was surgically induced in VE-Cre; E2F3(fl/fl) and E2F2-null (E2F2 KO) mice and their wild-type (WT) littermates, VE-Cre; E2F3(+/+) and E2F2 WT, respectively. The cardiac function, infarct size, and vascular density were significantly better in E2F2 KO mice and significantly worse in VE-Cre; E2F3(fl/fl) mice than in their WT littermates. The loss of E2F2 expression was associated with an increase in the proliferation of ECs both in vivo and in vitro, while the loss of E2F3 expression led to declines in EC proliferation. Thus, E2F3 promotes while E2F2 suppresses ischemic cardiac repair through corresponding changes in EC proliferation; and differential targeting of specific E2F members may provide a novel strategy for therapeutic angiogenesis of ischemic heart disease.

Lacidipine improves endothelial repair capacity of endothelial progenitor cells from patients with essential hypertension.

BACKGROUND: Endothelial progenitor cells (EPCs) play a critical role in maintaining the integrity of vascular endothelium following arterial injury. Lacidipine has a beneficial effect on endothelium of hypertensive patients, but limited data are available on EPCs-mediated endothelial protection. This study tests the hypothesis that lacidipine treatment can improve endothelial repair capacity of EPCs from hypertensive patients through increasing CXC chemokine receptor four (CXCR4) signaling. METHODS: In vivo reendothelialization capacity of EPCs from hypertensive patients with or without in vitro lacidipine treatment was examined in a nude mouse model of carotid artery injury. Expression of CXCR4 and alteration in migration and adhesion functions of EPCs were evaluated. RESULTS: Basal CXCR4 expression was markedly reduced in EPCs from hypertensive patients compared with normal subjects. In parallel, the phosphorylation of Janus kinase-2 (JAK-2) of EPCs, a CXCR4 downstream signaling, was also significantly decreased. Lacidipine promoted CXCR4/JAK-2 signaling expression of in vitro EPCs. Transplantation of EPCs pretreated with lacidipine significantly accelerated in vivo reendothelialization. The enhanced in vitro function and in vivo reendothelialization capacity of EPCs were inhibited by shRNA-mediated knockdown of CXCR4 expression or pretreatment with JAK-2 inhibitor AG490, respectively. In hypertensive patients, lacidipine treatment for 4 weeks also resulted in an upregulation of CXCR4/JAK-2 signaling of EPCs, which was associated with augmented EPCs-mediated reendothelialization and improved endothelial function. CONCLUSION: Deterioration of CXCR4 signaling may lead to impaired EPCs-mediated reendothelialization of hypertensive patients. Lacidipine-modified EPCs via a partially CXCR4 signaling contribute to enhanced endothelial repair capacity in hypertension.

Contrasting roles of E2F2 and E2F3 in endothelial cell growth and ischemic angiogenesis.

The growth of new blood vessels after ischemic injury requires endothelial cells (ECs) to divide and proliferate, and the E2F transcription factors are key regulators of the genes responsible for cell-cycle progression; however, the specific roles of individual E2Fs in ECs are largely unknown. To determine the roles of E2F2 and E2F3 in EC proliferation and the angiogenic response to ischemic injury, hind-limb ischemia was surgically induced in E2F2(-/-) mice, endothelial-specific E2F3-knockout (EndoE2F3(∆/∆)) mice, and their littermates with wild-type E2F2 and E2F3 expression. Two weeks later, Laser-Doppler perfusion measurements, capillary density, and endothelial proliferation were significantly greater in E2F2(-/-) mice and significantly lower in EndoE2F3(∆/∆) mice than in their littermates, and EndoE2F3(∆/∆) mice also developed toe and limb necrosis. The loss of E2F2 expression was associated with increases in the proliferation and G1/S-phase gene expression of isolated ECs, while the loss of E2F3 expression led to declines in these parameters. Thus E2F2 impairs, and endothelial E2F3 promotes, the angiogenic response to peripheral ischemic injury through corresponding changes in EC cell-cycle progression.

Regular exercise-induced increased number and activity of circulating endothelial progenitor cells attenuates age-related decline in arterial elasticity in healthy men.

BACKGROUND: Deficiency in number and activity of circulating EPCs is associated with reduced arterial elasticity in humans with advancing aging. Physical exercise can increase the number and activity of circulating EPCs in humans. Here we investigated whether regular exercise-induced enhanced circulating endothelial progenitor cells (EPCs) improves age-related decline in arterial elasticity in healthy men. METHODS: In a cross-sectional study, the number and activity of circulating EPCs as well as brachial-ankle pulse wave velocity (baPWV) of young and older sedentary or endurance-trained healthy men were studied. Then we observed the effect of regular exercise on circulating EPCs and baPWV of 10 older and 10 young sedentary healthy men. RESULTS: In both sedentary and endurance-trained men, the number and activity of circulating EPCs were significantly low in older men compared with young men, which was paralleled to increased baPWV. After three months of regular exercise, the number and activity of circulating EPCs increased, and the baPWV of 10 older and 10 young sedentary healthy men decreased. However, the increased number and activity of circulating EPCs and decreased baPWV of older sedentary healthy men were higher. There was a close correlation between circulating EPCs and baPWV. Multivariate analysis identified proliferative activity of circulating EPCs as an independent predictor of baPWV. CONCLUSIONS: The present study demonstrates for the first time that regular physical exercise-induced enhanced circulating EPCs attenuates age-related decline in arterial elasticity in healthy men. These findings provide novel insights into the protective effects of exercise on age-related vascular injury.

Shear stress-induced activation of Tie2-dependent signaling pathway enhances reendothelialization capacity of early endothelial progenitor cells.

Although endothelial progenitor cells (EPCs) play a pivotal role in the endothelial repair following arterial injury and shear stress has a beneficial effect on EPCs, however, the molecular mechanism underlying the influence of EPCs on the endothelial integrity and the regulation of shear stress on the EPC signaling remained to be studied. Here, we investigated the effects of laminar shear stress on the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2)-dependent signaling and its relation to in vivo reendothelialization capacity of human early EPCs. The human early EPCs were treated with shear stress. Shear stress in a dose-dependent manner increased angiopoietin-2 (Ang2)-induced migratory, adhesive and proliferatory activities of EPCs. Transplantation of EPCs treated by shear stress facilitated in vivo reendothelialization in nude mouse model of carotid artery injury. In parallel, the phosphorylation of Tie2 and Akt of EPCs in response to shear stress was significantly enhanced. With treatment of Tie2 knockdown or Akt inhibition, shear stress-induced phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) of EPCs was markedly suppressed. After Tie2/PI3K/Akt/eNOS signaling was blocked, the effects of shear stress on in vitro function and in vivo reendothelialization capacity of EPCs were significantly inhibited. The present findings demonstrate for the first time that Tie2/PI3k/Akt/eNOS signaling pathway is, at least in part, involved in the EPCs-mediated reendothelialization after arterial injury. The upregulation of shear stress-induced Tie2-dependent signaling contributes to enhanced in vivo reendothelialization capacity of human EPCs.

CXCR4 gene transfer contributes to in vivo reendothelialization capacity of endothelial progenitor cells.

AIMS: Endothelial progenitor cells (EPCs) play a pivotal role in endothelial repair after artery injury. The chemokine receptor CXCR4 is a key modulator of the homing of EPCs to impaired artery and reendothelialization. In this study, we addressed the hypothesis that CXCR4 gene transfer could enhance the reendothelialization capacity of EPCs. METHODS AND RESULTS: In vitro, human EPCs were expanded and transduced with adenovirus serotype 5 encoding the human CXCR4 gene (Ad5/CXCR4). In vitro, CXCR4 gene transfer augmented EPC migration and enhanced EPC adhesion to endothelial cell monolayers. Adhesion assays under flow conditions showed that CXCR4 gene transfer increased the ability of EPCs to arrest on fibronectin. To determine whether CXCR4 gene transfer facilitated therapeutic reendothelialization, the effect of EPCs on in vivo reendothelialization was examined in nude mice subjected to carotid artery injury. Compared with the vehicle, transplantation of EPCs with or without gene transfer significantly accelerated in vivo reendothelialization; however, transplantation of EPCs transduced with Ad5/CXCR4 had a further enhanced effect compared with control EPCs containing EPCs transduced with an adenovirus encoding enhanced green fluorescent protein gene or non-transduced EPCs. We also found that phosphorylation of Janus kinase-2 (JAK-2), a CXCR4 downstream signalling target, was increased in EPCs transduced with Ad5/CXCR4. The enhanced in vitro function and in vivo reendothelialization capacity of EPCs by CXCR4 gene transfer were abolished by neutralizing antibodies against CXCR4 or/and JAK-2 inhibitor AG490. CONCLUSION: The present study demonstrates that CXCR4 gene transfer contributes to the enhanced in vivo reendothelialization capacity of EPCs. Up-regulation of CXCR4 in human EPCs may become a novel therapeutic target for endothelial repair.