Bone marrow cells contribute to seven different endothelial cell populations in the heart.

Understanding the mechanisms underlying vascular regeneration in the heart is crucial for developing novel therapeutic strategies for myocardial ischemia. This study investigates the contribution of bone marrow-derived cells to endothelial cell populations in the heart, and their role in cardiac function and coronary circulation following repetitive ischemia (RI). Chimeric rats were created by transplanting BM cells from GFP female rats into irradiated male recipients. After engraftment chimeras were subjected to RI for 17 days. Vascular growth was assessed from recovery of cardiac function and increases in myocardial blood flow during LAD occlusion. After sorting GFP+ BM cells from heart and bone of Control and RI rats, single-cell RNA sequencing was implemented to determine the fate of BM cells. Our in vivo RI model demonstrated an improvement in cardiac function and myocardial blood flow after 17 days of RI with increased capillary density in the rats subjected to RI compared to Controls. Single-cell RNA sequencing of bone marrow cells isolated from rats' hearts identified distinct endothelial cell (EC) subpopulations. These ECs exhibited heterogeneous gene expression profiles and were enriched for markers of capillary, artery, lymphatic, venous, and immune ECs. Furthermore, BM-derived ECs in the RI group showed an angiogenic profile, characterized by upregulated genes associated with blood vessel development and angiogenesis. This study elucidates the heterogeneity of bone marrow-derived endothelial cells in the heart and their response to repetitive ischemia, laying the groundwork for targeting specific subpopulations for therapeutic angiogenesis in myocardial ischemia.

Takotsubo syndrome is a coronary microvascular disease: experimental evidence.

BACKGROUND AND AIMS: Takotsubo syndrome (TTS) is a conundrum without consensus about the cause. In a murine model of coronary microvascular dysfunction (CMD), abnormalities in myocardial perfusion played a key role in the development of TTS. METHODS AND RESULTS: Vascular Kv1.5 channels connect coronary blood flow to myocardial metabolism and their deletion mimics the phenotype of CMD. To determine if TTS is related to CMD, wild-type (WT), Kv1.5-/-, and TgKv1.5-/- (Kv1.5-/- with smooth muscle-specific expression Kv1.5 channels) mice were studied following transaortic constriction (TAC). Measurements of left ventricular (LV) fractional shortening (FS) in base and apex, and myocardial blood flow (MBF) were completed with standard and contrast echocardiography. Ribonucleic Acid deep sequencing was performed on LV apex and base from WT and Kv1.5-/- (control and TAC). Changes in gene expression were confirmed by real-time-polymerase chain reaction. MBF was increased with chromonar or by smooth muscle expression of Kv1.5 channels in the TgKv1.5-/-. TAC-induced systolic apical ballooning in Kv1.5-/-, shown as negative FS (P < 0.05 vs. base), which was not observed in WT, Kv1.5-/- with chromonar, or TgKv1.5-/-. Following TAC in Kv1.5-/-, MBF was lower in LV apex than in base. Increasing MBF with either chromonar or in TgKv1.5-/- normalized perfusion and function between LV apex and base (P = NS). Some genetic changes during TTS were reversed by chromonar, suggesting these were independent of TAC and more related to TTS. CONCLUSION: Abnormalities in flow regulation between the LV apex and base cause TTS. When perfusion is normalized between the two regions, normal ventricular function is restored.

Myocardial Blood Flow Control by Oxygen Sensing Vascular Kvß Proteins.

[Figure: see text].

The essential role for endothelial cell sprouting in coronary collateral growth.

RATIONALE: Coronary collateral growth is a natural bypass for ischemic heart diseases. It offers tremendous therapeutic benefit, but the process of coronary collateral growth isincompletely understood due to limited preclinical murine models that would enable interrogation of its mechanisms and processes via genetic modification and lineage tracing. Understanding the processes by which coronary collaterals develop can unlock new therapeutic strategies for ischemic heart disease. OBJECTIVE: To develop a murine model of coronary collateral growth by repetitive ischemia and investigate whether capillary endothelial cells could contribute to the coronary collateral formation in an adult mouse heart after repetitive ischemia by lineage tracing. METHODS AND RESULTS: A murine model of coronary collateral growth was developed using short episodes of repetitive ischemia. Repetitive ischemia stimulation resulted in robust collateral growth in adult mouse hearts, validated by high-resolution micro-computed tomography. Repetitive ischemia-induced collateral formation compensated ischemia caused by occlusion of the left anterior descending artery. Cardiac function improved during ischemia after repetitive ischemia, suggesting the improvement of coronary blood flow. A capillary-specific Cre driver (Apln-CreER) was used for lineage tracing capillary endothelial cells. ROSA mT/mG reporter mice crossed with the Apln-CreER transgene mice underwent a 17 days' repetitive ischemia protocol for coronary collateral growth. Two-photon and confocal microscopy imaging of heart slices revealed repetitive ischemia-induced coronary collateral growth initiated from sprouting Apelin+ endothelial cells. Newly formed capillaries in the collateral-dependent zone expanded in diameter upon repetitive ischemia stimulation and arterialized with smooth muscle cell recruitment, forming mature coronary arteries. Notably, pre-existing coronary arteries and arterioles were not Apelin+, and all Apelin+ collaterals arose from sprouting capillaries. Cxcr4, Vegfr2, Jag1, Mcp1, and Hif1⍺ mRNA levels in the repetitive ischemia-induced hearts were also upregulated at the early stage of coronary collateral growth, suggesting angiogenic signaling pathways are activated for coronary collaterals formation during repetitive ischemia. CONCLUSIONS: We developed a murine model of coronary collateral growth induced by repetitive ischemia. Our lineage tracing study shows that sprouting endothelial cells contribute to coronary collateral growth in adult mouse hearts. For the first time, sprouting angiogenesis is shown to give rise to mature coronary arteries in response to repetitive ischemia in the adult mouse hearts.

Mitochondrial DNA integrity and function are critical for endothelium-dependent vasodilation in rats with metabolic syndrome.

Endothelial dysfunction in diabetes is generally attributed to oxidative stress, but this view is challenged by observations showing antioxidants do not eliminate diabetic vasculopathy. As an alternative to oxidative stress-induced dysfunction, we interrogated if impaired mitochondrial function in endothelial cells is central to endothelial dysfunction in the metabolic syndrome. We observed reduced coronary arteriolar vasodilation to the endothelium-dependent dilator, acetylcholine (Ach), in Zucker Obese Fatty rats (ZOF, 34 ± 15% [mean ± standard deviation] 10-3 M) compared to Zucker Lean rats (ZLN, 98 ± 11%). This reduction in dilation occurred concomitantly with mitochondrial DNA (mtDNA) strand lesions and reduced mitochondrial complex activities in the endothelium of ZOF versus ZLN. To demonstrate endothelial dysfunction is linked to impaired mitochondrial function, administration of a cell-permeable, mitochondria-directed endonuclease (mt-tat-EndoIII), to repair oxidatively modified DNA in ZOF, restored mitochondrial function and vasodilation to Ach (94 ± 13%). Conversely, administration of a cell-permeable, mitochondria-directed exonuclease (mt-tat-ExoIII) produced mtDNA strand breaks in ZLN, reduced mitochondrial complex activities and vasodilation to Ach in ZLN (42 ± 16%). To demonstrate that mitochondrial function is central to endothelium-dependent vasodilation, we introduced (via electroporation) liver mitochondria (from ZLN) into the endothelium of a mesenteric vessel from ZOF and restored endothelium-dependent dilation to vasoactive intestinal peptide (VIP at 10-5 M, 4 ± 3% vasodilation before mitochondrial transfer and 48 ± 36% after transfer). Finally, to demonstrate mitochondrial function is key to endothelium-dependent dilation, we administered oligomycin (mitochondrial ATP synthase inhibitor) and observed a reduction in endothelium-dependent dilation. We conclude that mitochondrial function is critical for endothelium-dependent vasodilation.

Mechanism of the switch from NO to H2O2 in endothelium-dependent vasodilation in diabetes.

Coronary microvascular dysfunction is prevalent among people with diabetes and is correlated with cardiac mortality. Compromised endothelial-dependent dilation (EDD) is an early event in the progression of diabetes, but its mechanisms remain incompletely understood. Nitric oxide (NO) is the major endothelium-dependent vasodilatory metabolite in the healthy coronary circulation, but this switches to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a significant risk factor for CAD, we hypothesized that a similar NO-to-H2O2 switch would occur in diabetes. Vasodilation was measured ex vivo in isolated coronary arteries from wild type (WT) and microRNA-21 (miR-21) null mice on a chow or high-fat/high-sugar diet, and B6.BKS(D)-Leprdb/J (db/db) mice using myography. Myocardial blood flow (MBF), blood pressure, and heart rate were measured in vivo using contrast echocardiography and a solid-state pressure sensor catheter. RNA from coronary arteries, endothelial cells, and cardiac tissues was analyzed via quantitative real-time PCR for gene expression, and cardiac protein expression was assessed via western blot analyses. Superoxide was detected via electron paramagnetic resonance. (1) Ex vivo coronary EDD and in vivo MBF were impaired in diabetic mice. (2) Nω-Nitro-L-arginine methyl ester, an NO synthase inhibitor (L-NAME), inhibited ex vivo coronary EDD and in vivo MBF in WT. In contrast, polyethylene glycol-catalase, an H2O2 scavenger (Peg-Cat), inhibited diabetic mouse EDD ex vivo and MBF in vivo. (3) miR-21 was upregulated in diabetic mouse endothelial cells, and the deficiency of miR-21 prevented the NO-to-H2O2 switch and ameliorated diabetic mouse vasodilation impairments. (4) Diabetic mice displayed increased serum NO and H2O2, upregulated mRNA expression of Sod1, Sod2, iNos, and Cav1, and downregulated Pgc-1α in coronary arteries, but the deficiency of miR-21 reversed these changes. (5) miR-21-deficient mice exhibited increased cardiac PGC-1α, PPARα and eNOS protein and reduced endothelial superoxide. (6) Inhibition of PGC-1α changed the mRNA expression of genes regulated by miR-21, and overexpression of PGC-1α decreased the expression of miR-21 in high (25.5 mM) glucose treated coronary endothelial cells. Diabetic mice exhibit a NO-to-H2O2 switch in the mediator of coronary EDD, which contributes to microvascular dysfunction and is mediated by miR-21. This study represents the first mouse model recapitulating the NO-to-H2O2 switch seen in CAD patients in diabetes.

Reperfusion mediates heme impairment with increased protein cysteine sulfonation of mitochondrial complex III in the post-ischemic heart.

A serious consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which causes mitochondrial dysfunction. The cascading ROS can propagate and potentially induce heme bleaching and protein cysteine sulfonation (PrSO3H) of the mitochondrial electron transport chain. Herein we studied the mechanism of I/R-mediated irreversible oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the catalytic activity of complex III was significantly impaired. Spectroscopic analysis indicated that I/R mediated the destruction of hemes b and c + c1 in the mitochondria, supporting I/R-mediated complex III impairment. However, no significant impairment of complex III activity and heme damage were observed in mitochondria from the risk region of rat hearts subjected only to 30-min ischemia, despite a decreased state 3 respiration. In the I/R mitochondria, carbamidomethylated C122/C125 of cytochrome c1 via alkylating complex III with a down regulation of HCCS was exclusively detected, supporting I/R-mediated thioether defect of heme c1. LC-MS/MS analysis showed that I/R mitochondria had intensely increased complex III PrSO3H levels at the C236 ligand of the [2Fe2S] cluster of the Rieske iron­sulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSO3H of the hinge protein at C65 and of cytochrome c1 at C140 and C220, which are confined in the intermembrane space. MS analysis also showed that I/R extensively enhanced the PrSO3H of the core 1 (uqcrc1) and core 2 (uqcrc2) subunits in the matrix compartment, thus supporting the conclusion that complex III releases ROS to both sides of the inner membrane during reperfusion. Analysis of ischemic mitochondria indicated a modest reduction from the basal level of complex III PrSO3H detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSO3H. In conclusion, reperfusion-mediated heme damage with increased PrSO3H controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart.

Intracellular and exosomal microRNAome profiling of human vascular smooth muscle cells during replicative senescence.

Vascular aging is highly associated with cardiovascular morbidity and mortality. Although the senescence of vascular smooth muscle cells (VSMCs) has been well established as a major contributor to vascular aging, intracellular and exosomal microRNA (miRNA) signaling pathways in senescent VSMCs have not been fully elucidated. This study aimed to identify the differential expression of intracellular and exosomal miRNA in human VSMCs (hVSMCs) during replicative senescence. To achieve this aim, intracellular and exosomal miRNAs were isolated from hVSMCs and subsequently subjected to whole genome small RNA next-generation sequencing, bioinformatics analyses, and qPCR validation. Three significant findings were obtained. First, senescent hVSMC-derived exosomes tended to cluster together during replicative senescence and the molecular weight of the exosomal protein tumor susceptibility gene 101 (TSG-101) increased relative to the intracellular TSG-101, suggesting potential posttranslational modifications of exosomal TSG-101. Second, there was a significant decrease in both intracellular and exosomal hsa-miR-155-5p expression [n = 3, false discovery rate (FDR) < 0.05], potentially being a cell type-specific biomarker of hVSMCs during replicative senescence. Importantly, hsa-miR-155-5p was found to associate with cell-cycle arrest and elevated oxidative stress. Lastly, miRNAs from the intracellular pool, that is, hsa-miR-664a-3p, hsa-miR-664a-5p, hsa-miR-664b-3p, hsa-miR-4485-3p, hsa-miR-10527-5p, and hsa-miR-12136, and that from the exosomal pool, that is, hsa-miR-7704, were upregulated in hVSMCs during replicative senescence (n = 3, FDR < 0.05). Interestingly, these novel upregulated miRNAs were not functionally well annotated in hVSMCs to date. In conclusion, hVSMC-specific miRNA expression profiles during replicative senescence potentially provide valuable insights into the signaling pathways leading to vascular aging.NEW & NOTEWORTHY This is the first study on intracellular and exosomal miRNA profiling on human vascular smooth muscle cells during replicative senescence. Specific dysregulated sets of miRNAs were identified from human vascular smooth muscle cells. Hsa-miR-155-5p was significantly downregulated in both intracellular and exosomal hVSMCs, suggesting its crucial role in cellular senescence. Hsa-miR-155-5p might be the mediator in linking cellular senescence to vascular aging and atherosclerosis.

Exosomal microRNAs in the development of essential hypertension and its potential as biomarkers.

MicroRNAs (miRNAs) are small regulatory molecules that are involved in posttranscriptional modifications. These noncoding RNAs are usually ferried by extracellular carriers such as exosomes or other protein and lipid carriers inside a range of body fluids including plasma and urine. Due to their ability to withstand harsh external conditions, exosomal miRNAs possess enormous potential as noninvasive disease biomarkers for, notably hypertension, whereby exosomal miRNAs have been implicated in its pathophysiological processes. More importantly, alterations in the microenvironment as a result of disease progression can induce active and selective loading of miRNAs into exosomes. In this paper, we first review the mechanisms of miRNA loading into exosomes, followed by the roles of exosomal miRNAs in the development of hypertension, and the potentials of exosomal miRNAs as biomarkers in comparison with other free circulating miRNAs. Finally, challenges and future research surrounding exosomal miRNAs will also be discussed. This review will aid in the understanding of noninvasive biomarkers for the early diagnosis of hypertension and for probing therapeutic efficacy.

MicroRNA regulation of vascular smooth muscle cells and its significance in cardiovascular diseases.

Cardiovascular disease (CVD) is among the leading causes of death worldwide. MicroRNAs (miRNAs), regulatory molecules that repress protein expression, have attracted considerable attention in CVD research. The vasculature plays a big role in CVD development and progression and dysregulation of vascular cells underlies the root of many vascular diseases. This review provides a brief introduction of the biogenesis of miRNAs and exosomes, followed by overview of the regulatory mechanisms of miRNAs in vascular smooth muscle cells (VSMCs) intracellular signaling during phenotypic switching, senescence, calcification, and neointimal hyperplasia. Evidence of extracellular signaling of VSMCs and other cells via exosomal and circulating miRNAs is also presented. Lastly, current drawbacks and limitations of miRNA studies in CVD research and potential ways to overcome these disadvantages are discussed in detail. In-depth understanding of VSMC regulation via miRNAs will add substantial knowledge and advance research in diagnosis, disease progression, and (or) miRNA-derived therapeutic approaches in CVD research.

TRPV4 deletion protects heart from myocardial infarction-induced adverse remodeling via modulation of cardiac fibroblast differentiation.

Cardiac fibrosis caused by adverse cardiac remodeling following myocardial infarction can eventually lead to heart failure. Although the role of soluble factors such as TGF-ß is well studied in cardiac fibrosis following myocardial injury, the physiological role of mechanotransduction is not fully understood. Here, we investigated the molecular mechanism and functional role of TRPV4 mechanotransduction in cardiac fibrosis. TRPV4KO mice, 8 weeks following myocardial infarction (MI), exhibited preserved cardiac function compared to WT mice. Histological analysis demonstrated reduced cardiac fibrosis in TRPV4KO mice. We found that WT CF exhibited hypotonicity-induced calcium influx and extracellular matrix (ECM)-stiffness-dependent differentiation in response to TGF-ß1. In contrast, TRPV4KO CF did not display hypotonicity-induced calcium influx and failed to differentiate on high-stiffness ECM gels even in the presence of saturating amounts of TGF-ß1. Mechanistically, TRPV4 mediated cardiac fibrotic gene promoter activity and fibroblast differentiation through the activation of the Rho/Rho kinase pathway and the mechanosensitive transcription factor MRTF-A. Our findings suggest that genetic deletion of TRPV4 channels protects heart from adverse cardiac remodeling following MI by modulating Rho/MRTF-A pathway-mediated cardiac fibroblast differentiation and cardiac fibrosis.

Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4.

VEGF signaling via VEGF receptor-2 (VEGFR2) is a major regulator of endothelial cell (EC) functions, including angiogenesis. Although most studies of angiogenesis focus on soluble VEGF signaling, mechanical signaling also plays a critical role. Here, we examined the consequence of disruption of mechanical signaling on soluble signaling pathways. Specifically, we observed that small interfering RNA (siRNA) knockdown of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), significantly reduced perinuclear (Golgi) VEGFR2 in human ECs with a concomitant increase in phosphorylation at Y1175 and membrane translocation. TRPV4 knockout (KO) ECs exhibited increased plasma membrane localization of phospho-VEGFR2 compared with normal ECs. The knockdown also increased phospho-VEGFR2 in whole cell lysates and membrane fractions compared with control siRNA-treated cells. siRNA knockdown of TRPV4 enhanced nuclear localization of mechanosensitive transcription factors, yes-associated protein/transcriptional coactivator with PDZ-binding motif via rho kinase, which were shown to increase VEGFR2 trafficking to the plasma membrane. Furthermore, TRPV4 deletion/knockdown enhanced VEGF-mediated migration in vitro and increased expression of VEGFR2 in vivo in the vasculature of TRPV4 KO tumors compared with wild-type tumors. Our results thus show that TRPV4 channels regulate VEGFR2 trafficking and activation to identify novel cross-talk between mechanical (TRPV4) and soluble (VEGF) signaling that controls EC migration and angiogenesis.-Kanugula, A. K., Adapala, R. K., Midha, P., Cappelli, H. C., Meszaros, J. G., Paruchuri, S., Chilian, W. M., Thodeti, C. K., Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4.

Vago-Splenic Axis in Signal Transduction of Remote Ischemic Preconditioning in Pigs and Rats.

RATIONALE: The signal transduction of remote ischemic conditioning is still largely unknown. OBJECTIVE: Characterization of neurohumoral signal transfer and vago-splenic axis in remote ischemic preconditioning (RIPC). METHODS AND RESULTS: Anesthetized pigs were subjected to 60 minutes of coronary occlusion and 180 minutes of reperfusion (placebo+ischemia/reperfusion [PLA+I/R]). RIPC was induced by 4×5/5 minutes of hindlimb I/R 90 minutes before coronary occlusion (RIPC+I/R). Arterial blood samples were taken after placebo or RIPC before I/R. In subgroups of pigs, bilateral cervical vagotomy, splenectomy, or splenic denervation were performed before PLA+I/R or RIPC+I/R, respectively. In pigs with RIPC+I/R, infarct size (percentage of area at risk) was less than in those with PLA+I/R (23±12% versus 45±8%); splenectomy or splenic denervation abrogated (splenectomy+RIPC+I/R: 38±15%; splenic denervation+RIPC+I/R: 43±5%), and vagotomy attenuated (vagotomy+RIPC+I/R: 36±11%) RIPC protection. RIPC increased phosphorylation of STAT3 (signal transducer and activator of transcription 3) in left ventricular biopsies taken at early reperfusion. Splenectomy or splenic denervation, but not vagotomy, abolished this increased phosphorylation. In rats with vagotomy, splenectomy, or splenic denervation, RIPC (3×5/5 minutes of hindlimb occlusion/reperfusion) or placebo was performed, respectively. Hearts were isolated, saline perfused, and subjected to 30/120-minute global I/R. With RIPC, infarct size (percentage of ventricular mass) was less (20±7%) than with placebo (37±6%), and vagotomy, splenectomy, or splenic denervation abrogated RIPC protection (38±12%, 36±9%, and 36±7%), respectively. Rat spleens were isolated, saline perfused, and splenic effluate (SEff) was sampled after infusion with carbachol (SEffcarbachol) or saline (SEffsaline). Pig plasma or SEff was infused into isolated perfused rat hearts subjected to global I/R. Infarct size was less with infusion of RIPC+I/Rplasma+ (24±6%) than with PLA+I/Rplasma (40±8%), vagotomy+PLA+I/Rplasma (39±11%), splenectomy+PLA+I/Rplasma (35±8%), vagotomy+RIPC+I/Rplasma (40±9%), splenectomy+RIPC+I/Rplasma (33±9%), or splenic denervation+RIPC+I/Rplasma (39±8%), respectively. With infusion of SEffcarbachol, infarct size was less than with infusion of SEffsaline (24 [19-27]% versus 35 [32-38]%). CONCLUSIONS: Activation of a vago-splenic axis is causally involved in RIPC cardioprotection.

Ischemic Heart Disease Pathophysiology Paradigms Overview: From Plaque Activation to Microvascular Dysfunction.

Ischemic heart disease still represents a large burden on individuals and health care resources worldwide. By conventions, it is equated with atherosclerotic plaque due to flow-limiting obstruction in large-medium sized coronary arteries. However, clinical, angiographic and autoptic findings suggest a multifaceted pathophysiology for ischemic heart disease and just some cases are caused by severe or complicated atherosclerotic plaques. Currently there is no well-defined assessment of ischemic heart disease pathophysiology that satisfies all the observations and sometimes the underlying mechanism to everyday ischemic heart disease ward cases is misleading. In order to better examine this complicated disease and to provide future perspectives, it is important to know and analyze the pathophysiological mechanisms that underline it, because ischemic heart disease is not always determined by atherosclerotic plaque complication. Therefore, in order to have a more complete comprehension of ischemic heart disease we propose an overview of the available pathophysiological paradigms, from plaque activation to microvascular dysfunction.

Cardioprotection during ischemia by coronary collateral growth.

Ischemic heart diseases (IHD) cause millions of deaths around the world annually. While surgical and pharmacological interventions are commonly used to treat patients with IHD, their efficacy varies from patient to patient and is limited by the severity of the disease. One promising, at least theoretically, approach for treating IHD is induction of coronary collateral growth (CCG). Coronary collaterals are arteriole-to-arteriole anastomoses that can undergo expansion and remodeling in the setting of coronary disease when the disease elicits myocardial ischemia and creates a pressure difference across the collateral vessel that creates unidirectional flow. Well-developed collaterals can restore blood flow in the ischemic area of the myocardium and protect the myocardium at risk. Moreover, such collaterals are correlated to reduced mortality and infarct size and better cardiac function during occlusion of coronary arteries. Therefore, understanding the process of CCG is highly important as a potentially viable treatment of IHD. While there are several excellent review articles on this topic, this review will provide a unified overview of the various aspects related to CCG as well as an update of the advancements in the field. We also call for more detailed studies with an interdisciplinary approach to advance our knowledge of CCG. In this review, we will describe growth of coronary collaterals, the various factors that contribute to CCG, animal models used to study CCG, and the cardioprotective effects of coronary collaterals during ischemia. We will also discuss the impairment of CCG in metabolic syndrome and the therapeutic potentials of CCG in IHD.

Exosomes derived from induced vascular progenitor cells promote angiogenesis in vitro and in an in vivo rat hindlimb ischemia model.

Induced vascular progenitor cells (iVPCs) were created as an ideal cell type for regenerative medicine and have been reported to positively promote collateral blood flow and improve cardiac function in a rat model of myocardial ischemia. Exosomes have emerged as a novel biomedicine that mimics the function of the donor cells. We investigated the angiogenic activity of exosomes from iPVCs (iVPC-Exo) as a cell-free therapeutic approach for ischemia. Exosomes from iVPCs and rat aortic endothelial cells (RAECs) were isolated using a combination of ultrafiltration and size-exclusion chromatography. Nanoparticle tracking analysis revealed that exosome isolates fell within the exosomal diameter (<150 nm). These exosomes contained known markers Alix and TSG101, and their morphology was validated using transmission electron microscopy. When compared with RAECs, iVPCs significantly increased the secretion of exosomes. Cardiac microvascular endothelial cells and aortic ring explants were pretreated with RAEC-Exo or iVPC-Exo, and basal medium was used as a control. iVPC-Exo exerted an in vitro angiogenic effect on the proliferation, tube formation, and migration of endothelial cells and stimulated microvessel sprouting in an ex vivo aortic ring assay. Additionally, iVPC-Exo increased blood perfusion in a hindlimb ischemia model. Proangiogenic proteins (pentraxin-3 and insulin-like growth factor-binding protein-3) and microRNAs (-143-3p, -291b, and -20b-5p) were found to be enriched in iVPC-Exo, which may mediate iVPC-Exo induced vascular growth. Our findings demonstrate that treatment with iVPC-Exo promotes angiogenesis in vitro, ex vivo, and in vivo. Collectively, these findings indicate a novel cell-free approach for therapeutic angiogenesis.NEW & NOTEWORTHY The results of this work demonstrate exosomes as a novel physiological mechanism by which induced vascular progenitor cells exert their angiogenic effect. Moreover, angiogenic cargo of proteins and microRNAs may define the biological contributors in activating endothelial cells to form a new capillary plexus for ischemic vascular diseases.

Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling.

BACKGROUND: Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. METHODS: Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×108 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or KATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease. RESULTS: Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide and prostaglandins. CONCLUSIONS: Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP, which can act through a diverse portfolio of purinergic signaling pathways. These events can reverse hindlimb ischemia in mice for >24 hours and increase muscle blood flow in patients with sickle cell disease. CLINICAL TRIAL REGISTRATION: URL: http://clinicaltrials.gov. Unique identifier: NCT01566890.

Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association.

Reactive oxygen species and reactive nitrogen species are biological molecules that play important roles in cardiovascular physiology and contribute to disease initiation, progression, and severity. Because of their ephemeral nature and rapid reactivity, these species are difficult to measure directly with high accuracy and precision. In this statement, we review current methods for measuring these species and the secondary products they generate and suggest approaches for measuring redox status, oxidative stress, and the production of individual reactive oxygen and nitrogen species. We discuss the strengths and limitations of different methods and the relative specificity and suitability of these methods for measuring the concentrations of reactive oxygen and reactive nitrogen species in cells, tissues, and biological fluids. We provide specific guidelines, through expert opinion, for choosing reliable and reproducible assays for different experimental and clinical situations. These guidelines are intended to help investigators and clinical researchers avoid experimental error and ensure high-quality measurements of these important biological species.

The JCR:LA-cp rat: a novel rodent model of cystic medial necrosis.

Although there are multiple rodent models of the metabolic syndrome, very few develop vascular complications. In contrast, the JCR:LA-cp rat develops both metabolic syndrome and early atherosclerosis in predisposed areas. However, the pathology of the normal vessel wall has not been described. We examined JCR:LA control (+/+) or cp/cp rats fed normal chow diet for 6 or 18 mo. JCR:LA-cp rats developed multiple features of advanced cystic medial necrosis including "cysts," increased collagen formation and proteoglycan deposition around cysts, apoptosis of vascular smooth muscle cells, and spotty medial calcification. These appearances began within 6 mo and were extensive by 18 mo. JCR:LA-cp rats had reduced medial cellularity, increased medial thickness, and vessel hypoxia that was most marked in the adventitia. In conclusion, the normal chow-fed JCR:LA-cp rat represents a novel rodent model of cystic medial necrosis, associated with multiple metabolic abnormalities, vascular smooth muscle cell apoptosis, and vessel hypoxia.NEW & NOTEWORTHY Triggers for cystic medial necrosis (CMN) have been difficult to study due to lack of animal models to recapitulate the pathologies seen in humans. Our study is the first description of CMN in the rat. Thus the JCR:LA-cp rat represents a useful model to investigate the underlying molecular changes leading to the development of CMN.

Kv1.3 channels facilitate the connection between metabolism and blood flow in the heart.

The connection between metabolism and flow in the heart, metabolic dilation, is essential for cardiac function. We recently found redox-sensitive Kv1.5 channels play a role in coronary metabolic dilation; however, more than one ion channel likely plays a role in this process as animals null for these channels still showed limited coronary metabolic dilation. Accordingly, we examined the role of another Kv1 family channel, the energetically linked Kv1.3 channel, in coronary metabolic dilation. We measured myocardial blood flow (contrast echocardiography) during norepinephrine-induced increases in cardiac work (heart rate x mean arterial pressure) in WT, WT mice given correolide (preferential Kv1.3 antagonist), and Kv1.3-null mice (Kv1.3-/- ). We also measured relaxation of isolated small arteries mounted in a myograph. During increased cardiac work, myocardial blood flow was attenuated in Kv1.3-/- and in correolide-treated mice. In isolated vessels from Kv1.3-/- mice, relaxation to H2 O2 was impaired (vs WT), but responses to adenosine and acetylcholine were equivalent to WT. Correolide reduced dilation to adenosine and acetylcholine in WT and Kv1.3-/- , but had no effect on H2 O2 -dependent dilation in vessels from Kv1.3-/- mice. We conclude that Kv1.3 channels participate in the connection between myocardial blood flow and cardiac metabolism.