mouse-model-of-coronary-collateral-growth-through-repetitive-ischemia

Mouse Model of Coronary Collateral Growth Through Repetitive Ischemia

Coronary collaterals are a natural bypass in ischemic heart diseases (IHD), and so for many years, coronary collateral growth (CCG) has been a promising ...

Northeast Ohio Medical University

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.

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

Our goal was to determine the mechanism by which mitochondrial oxidative stress impairs collateral growth in the heart.

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

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.

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

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.

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

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 H O 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 H O -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.

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

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.

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

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.

Cardioprotection during ischemia by coronary collateral growth.

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 (HO) in coronary artery disease (CAD) patients. Because diabetes is a significant risk factor for CAD, we hypothesized that a similar NO-to-HO 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)-Lepr/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 HO 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-HO switch and ameliorated diabetic mouse vasodilation impairments. (4) Diabetic mice displayed increased serum NO and HO, 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-HO 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-HO switch seen in CAD patients in diabetes.

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

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.

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

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.

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

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.

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

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.

Is miR-21 A Therapeutic Target in Cardiovascular Disease?

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.

Article	Year
Induction of vascular progenitor cells from endothelial cells stimulates coronary collateral growth. Circulation research\| PubMed ID: 22095729	2012
Mitochondrial oxidative stress corrupts coronary collateral growth by activating adenosine monophosphate activated kinase-α signaling. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 23788766	2013
Novel thiazolidinedione mitoNEET ligand-1 acutely improves cardiac stem cell survival under oxidative stress. Basic research in cardiology\| PubMed ID: 25725808	2015
Requisite Role of Kv1.5 Channels in Coronary Metabolic Dilation. Circulation research\| PubMed ID: 26224794	2015
Kv1.3 channels facilitate the connection between metabolism and blood flow in the heart. Microcirculation (New York, N.Y. : 1994)\| PubMed ID: 28504408	2017
Alignment of inducible vascular progenitor cells on a micro-bundle scaffold improves cardiac repair following myocardial infarction. Basic research in cardiology\| PubMed ID: 28540527	2017
Cardioprotection during ischemia by coronary collateral growth. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 30379567	2019
Mechanism of the switch from NO to HO in endothelium-dependent vasodilation in diabetes. Basic research in cardiology\| PubMed ID: 35024970	2022
The essential role for endothelial cell sprouting in coronary collateral growth. Journal of molecular and cellular cardiology\| PubMed ID: 35074317	2022
Role of endothelial CXCR4 in the development of aortic valve stenosis. Frontiers in cardiovascular medicine\| PubMed ID: 36148060	2022
The Roles of Bone Marrow-Derived Stem Cells in Coronary Collateral Growth Induced by Repetitive Ischemia. Cells\| PubMed ID: 36672176	2023
Is miR-21 A Therapeutic Target in Cardiovascular Disease? International journal of drug discovery and pharmacology\| PubMed ID: 37799562	2023
Bone marrow cells contribute to seven different endothelial cell populations in the heart. Basic research in cardiology\| PubMed ID: 38963562	2024

Molly Enrick

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