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.

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.

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.

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.

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.

Alignment of inducible vascular progenitor cells on a micro-bundle scaffold improves cardiac repair following myocardial infarction.

Ischemic heart disease is still the leading cause of death even with the advancement of pharmaceutical therapies and surgical procedures. Early vascularization in the ischemic heart is critical for a better outcome. Although stem cell therapy has great potential for cardiovascular regeneration, the ideal cell type and delivery method of cells have not been resolved. We tested a new approach of stem cell therapy by delivery of induced vascular progenitor cells (iVPCs) grown on polymer micro-bundle scaffolds in a rat model of myocardial infarction. iVPCs partially reprogrammed from vascular endothelial cells (ECs) had potent angiogenic potential and were able to simultaneously differentiate into vascular smooth muscle cells (SMCs) and ECs in 2D culture. Under hypoxic conditions, iVPCs also secreted angiogenic cytokines such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as measured by enzyme-linked immunosorbent assay (ELISA). A longitudinal micro-scaffold made from poly(lactic-co-glycolic acid) was sufficient for the growth and delivery of iVPCs. Co-cultured ECs and SMCs aligned well on the micro-bundle scaffold similarly as in the vessels. 3D cell/polymer micro-bundles formed by iVPCs and micro-scaffolds were transplanted into the ischemic myocardium in a rat model of myocardial infarction (MI) with ligation of the left anterior descending artery. Our in vivo data showed that iVPCs on the micro-bundle scaffold had higher survival, and better retention and engraftment in the myocardium than free iVPCs. iVPCs on the micro-bundles promoted better cardiomyocyte survival than free iVPCs. Moreover, iVPCs and iVPC/polymer micro-bundles treatment improved cardiac function (ejection fraction and fractional shortening, endocardial systolic volume) measured by echocardiography, increased vessel density, and decreased infarction size [endocardial and epicardial infarct (scar) length] better than untreated controls at 8 weeks after MI. We conclude that iVPCs grown on a polymer micro-bundle scaffold are new promising approach for cell-based therapy designed for cardiovascular regeneration in ischemic heart disease.

Requisite Role of Kv1.5 Channels in Coronary Metabolic Dilation.

RATIONALE: In the working heart, coronary blood flow is linked to the production of metabolites, which modulate tone of smooth muscle in a redox-dependent manner. Voltage-gated potassium channels (Kv), which play a role in controlling membrane potential in vascular smooth muscle, have certain members that are redox-sensitive. OBJECTIVE: To determine the role of redox-sensitive Kv1.5 channels in coronary metabolic flow regulation. METHODS AND RESULTS: In mice (wild-type [WT], Kv1.5 null [Kv1.5(-/-)], and Kv1.5(-/-) and WT with inducible, smooth muscle-specific expression of Kv1.5 channels), we measured mean arterial pressure, myocardial blood flow, myocardial tissue oxygen tension, and ejection fraction before and after inducing cardiac stress with norepinephrine. Cardiac work was estimated as the product of mean arterial pressure and heart rate. Isolated arteries were studied to establish whether genetic alterations modified vascular reactivity. Despite higher levels of cardiac work in the Kv1.5(-/-) mice (versus WT mice at baseline and all doses of norepinephrine), myocardial blood flow was lower in Kv1.5(-/-) mice than in WT mice. At high levels of cardiac work, tissue oxygen tension dropped significantly along with ejection fraction. Expression of Kv1.5 channels in smooth muscle in the null background rescued this phenotype of impaired metabolic dilation. In isolated vessels from Kv1.5(-/-) mice, relaxation to H2O2 was impaired, but responses to adenosine and acetylcholine were normal compared with those from WT mice. CONCLUSIONS: Kv1.5 channels in vascular smooth muscle play a critical role in coupling myocardial blood flow to cardiac metabolism. Absence of these channels disassociates metabolism from flow, resulting in cardiac pump dysfunction and tissue hypoxia.

Novel thiazolidinedione mitoNEET ligand-1 acutely improves cardiac stem cell survival under oxidative stress.

Ischemic heart disease (IHD) is a leading cause of death worldwide, and regenerative therapies through exogenous stem cell delivery hold promising potential. One limitation of such therapies is the vulnerability of stem cells to the oxidative environment associated with IHD. Accordingly, manipulation of stem cell mitochondrial metabolism may be an effective strategy to improve survival of stem cells under oxidative stress. MitoNEET is a redox-sensitive, mitochondrial target of thiazolidinediones (TZDs), and influences cellular oxidative capacity. Pharmacological targeting of mitoNEET with the novel TZD, mitoNEET Ligand-1 (NL-1), improved cardiac stem cell (CSC) survival compared to vehicle (0.1% DMSO) during in vitro oxidative stress (H2O2). 10 µM NL-1 also reduced CSC maximal oxygen consumption rate (OCR) compared to vehicle. Following treatment with dexamethasone, CSC maximal OCR increased compared to baseline, but NL-1 prevented this effect. Smooth muscle α-actin expression increased significantly in CSC following differentiation compared to baseline, irrespective of NL-1 treatment. When CSCs were treated with glucose oxidase for 7 days, NL-1 significantly improved cell survival compared to vehicle (trypan blue exclusion). NL-1 treatment of cells isolated from mitoNEET knockout mice did not increase CSC survival with H2O2 treatment. Following intramyocardial injection of CSCs into Zucker obese fatty rats, NL-1 significantly improved CSC survival after 24 h, but not after 10 days. These data suggest that pharmacological targeting of mitoNEET with TZDs may acutely protect stem cells following transplantation into an oxidative environment. Continued treatment or manipulation of mitochondrial metabolism may be necessary to produce long-term benefits related to stem cell therapies.

Induction of vascular progenitor cells from endothelial cells stimulates coronary collateral growth.

RATIONALE: A well-developed coronary collateral circulation improves the morbidity and mortality of patients following an acute coronary occlusion. Although regenerative medicine has great potential in stimulating vascular growth in the heart, to date there have been mixed results, and the ideal cell type for this therapy has not been resolved. OBJECTIVE: To generate induced vascular progenitor cells (iVPCs) from endothelial cells, which can differentiate into vascular smooth muscle cells (VSMCs) or endothelial cells (ECs), and test their capability to stimulate coronary collateral growth. METHODS AND RESULTS: We reprogrammed rat ECs with the transcription factors Oct4, Klf4, Sox2, and c-Myc. A population of reprogrammed cells was derived that expressed pluripotent markers Oct4, SSEA-1, Rex1, and AP and hemangioblast markers CD133, Flk1, and c-kit. These cells were designated iVPCs because they remained committed to vascular lineage and could differentiate into vascular ECs and VSMCs in vitro. The iVPCs demonstrated better in vitro angiogenic potential (tube network on 2-dimensional culture, tube formation in growth factor reduced Matrigel) than native ECs. The risk of teratoma formation in iVPCs is also reduced in comparison with fully reprogrammed induced pluripotent stem cells (iPSCs). When iVPCs were implanted into myocardium, they engrafted into blood vessels and increased coronary collateral flow (microspheres) and improved cardiac function (echocardiography) better than iPSCs, mesenchymal stem cells, native ECs, and sham treatments. CONCLUSIONS: We conclude that iVPCs, generated by partially reprogramming ECs, are an ideal cell type for cell-based therapy designed to stimulate coronary collateral growth.

Mitochondrial oxidative stress corrupts coronary collateral growth by activating adenosine monophosphate activated kinase-α signaling.

OBJECTIVE: Our goal was to determine the mechanism by which mitochondrial oxidative stress impairs collateral growth in the heart. APPROACH AND RESULTS: Rats were treated with rotenone (mitochondrial complex I inhibitor that increases reactive oxygen species production) or sham-treated with vehicle and subjected to repetitive ischemia protocol for 10 days to induce coronary collateral growth. In control rats, repetitive ischemia increased flow to the collateral-dependent zone; however, rotenone treatment prevented this increase suggesting that mitochondrial oxidative stress compromises coronary collateral growth. In addition, rotenone also attenuated mitochondrial complex I activity and led to excessive mitochondrial aggregation. To further understand the mechanistic pathway(s) involved, human coronary artery endothelial cells were treated with 50 ng/mL vascular endothelial growth factor, 1 µmol/L rotenone, and rotenone/vascular endothelial growth factor for 48 hours. Vascular endothelial growth factor induced robust tube formation; however, rotenone completely inhibited this effect (P<0.05 rotenone versus vascular endothelial growth factor treatment). Inhibition of tube formation by rotenone was also associated with significant increase in mitochondrial superoxide generation. Immunoblot analyses of human coronary artery endothelial cells with rotenone treatment showed significant activation of adenosine monophosphate activated kinase (AMPK)-α and inhibition of mammalian target of rapamycin and p70 ribosomal S6 kinase. Activation of AMPK-α suggested impairments in energy production, which was reflected by decrease in O2 consumption and bioenergetic reserve capacity of cultured cells. Knockdown of AMPK-α (siRNA) also preserved tube formation during rotenone, suggesting the negative effects were mediated by the activation of AMPK-α. Conversely, expression of a constitutively active AMPK-α blocked tube formation. CONCLUSIONS: We conclude that activation of AMPK-α during mitochondrial oxidative stress inhibits mammalian target of rapamycin signaling, which impairs phenotypic switching necessary for the growth of blood vessels.

Is miR-21 A Therapeutic Target in Cardiovascular Disease?

microRNA-21 (miR-21) serves a multitude of functions at the molecular level through its regulation of messenger RNA. Previous research has sparked interest in the role of miR-21 as a potential therapeutic target in cardiovascular diseases. miR-21 expression contributes to the differentiation, proliferation, and maturation of many cell types, such as fibroblasts, endothelial cells, cardiomyocytes, and endothelial progenitor cells. The function of miR-21 depends upon its expression level in the specific cell types and downstream targets, which determine cell fate. Under pathological conditions, the expression level of miR-21 is altered, leading to abnormal gene regulation of downstream signaling and cardiovascular diseases such as hypertension, cardiac hypertrophy and fibrosis, atherosclerosis, and heart failure. Agomirs or antagomirs can be introduced into the respective tissue type to reverse or stop the progression of the disease. Exosomes in the extracellular vesicles, which mediate many cellular events with high biocompatibility, have a high potential of efficiently delivering miR-21 to their targeted cells. The critical role of miR-21 in cardiovascular disease (CVD) is indisputable, but there are controversial reports on the function of miR-21 in the same disease. This discrepancy sparks interest in better understanding the role of miR-21 in different tissues under different stages of various diseases and the mechanism of how miR-21 inhibitors work.

The Roles of Bone Marrow-Derived Stem Cells in Coronary Collateral Growth Induced by Repetitive Ischemia.

Many clinical trials have attempted to use stem cells to treat ischemic heart diseases (IHD), but the benefits have been modest. Though coronary collaterals can be a "natural bypass" for IHD patients, the regulation of coronary collateral growth (CCG) and the role of endogenous stem cells in CCG are not fully understood. In this study, we used a bone marrow transplantation scheme to study the role of bone marrow stem cells (BMSCs) in a rat model of CCG. Transgenic GFP rats were used to trace BMSCs after transplantation; GFP bone marrow was harvested or sorted for bone marrow transplantation. After recovering from transplantation, the recipient rats underwent 10 days of repetitive ischemia (RI), with echocardiography before and after RI, to measure cardiac function and myocardial blood flow. At the end of RI, the rats were sacrificed for the collection of bone marrow for flow cytometry or heart tissue for imaging analysis. Our study shows that upon RI stimulation, BMSCs homed to the recipient rat hearts' collateral-dependent zone (CZ), proliferated, differentiated into endothelial cells, and engrafted in the vascular wall for collateral growth. These RI-induced collaterals improved coronary blood flow and cardiac function in the recipients' hearts during ischemia. Depletion of donor CD34+ BMSCs led to impaired CCG in the recipient rats, indicating that this cell population is essential to the process. Overall, these results show that BMSCs contribute to CCG and suggest that regulation of the function of BMSCs to promote CCG might be a potential therapeutic approach for IHD.

Role of endothelial CXCR4 in the development of aortic valve stenosis.

Background: CXCL12/CXCR4 signaling is essential in cardiac development and repair, however, its contribution to aortic valve stenosis (AVS) remains unclear. In this study, we tested the role of endothelial CXCR4 on the development of AVS. Materials and methods: We generated CXCR4 endothelial cell-specific knockout mice (EC CXCR4 KO) by crossing CXCR4fl/fl mice with Tie2-Cre mice to study the role of endothelial cell CXCR4 in AVS. CXCR4fl/fl mice were used as controls. Echocardiography was used to assess the aortic valve and cardiac function. Heart samples containing the aortic valve were stained using Alizarin Red for detection of calcification. Masson's trichrome staining was used for the detection of fibrosis. The apex of the heart samples was stained with wheat germ agglutinin (WGA) to visualize ventricular hypertrophy. Results: Compared with the control group, the deletion of CXCR4 in endothelial cells led to significantly increased aortic valve peak velocity and aortic valve peak pressure gradient, with decreased aortic valve area and ejection fraction. EC CXCR4 KO mice also developed cardiac hypertrophy as evidenced by increased diastolic and systolic left ventricle posterior wall thickness (LVPW), cardiac myocyte size, and heart weight (HW) to body weight (BW) ratio. Our data also confirmed increased microcalcifications, interstitial fibrosis, and thickened valvular leaflets of the EC CXCR4 KO mice. Conclusion: The data collected throughout this study suggest the deletion of CXCR4 in endothelial cells is linked to the development of aortic valve stenosis and left ventricular hypertrophy. The statistically significant parameters measured indicate that endothelial cell CXCR4 plays an important role in aortic valve development and function. We have compiled compelling evidence that EC CXCR4 KO mice can be used as a novel model for AVS.

Centriolar SAS-7 acts upstream of SPD-2 to regulate centriole assembly and pericentriolar material formation.

The centriole/basal body is a eukaryotic organelle that plays essential roles in cell division and signaling. Among five known core centriole proteins, SPD-2/Cep192 is the first recruited to the site of daughter centriole formation and regulates the centriolar localization of the other components in C. elegans and in humans. However, the molecular basis for SPD-2 centriolar localization remains unknown. Here, we describe a new centriole component, the coiled-coil protein SAS-7, as a regulator of centriole duplication, assembly and elongation. Intriguingly, our genetic data suggest that SAS-7 is required for daughter centrioles to become competent for duplication, and for mother centrioles to maintain this competence. We also show that SAS-7 binds SPD-2 and regulates SPD-2 centriolar recruitment, while SAS-7 centriolar localization is SPD-2-independent. Furthermore, pericentriolar material (PCM) formation is abnormal in sas-7 mutants, and the PCM-dependent induction of cell polarity that defines the anterior-posterior body axis frequently fails. We conclude that SAS-7 functions at the earliest step in centriole duplication yet identified and plays important roles in the orchestration of centriole and PCM assembly.