Coronary Collateral Growth Research Articles

Asymmetric Dimethylarginine (ADMA) in Cardiovascular Disease, Cardiac Ischemia/reperfusion Injury, and Ischemic Non-obstructive Coronary Artery Disease: Biochemical and Pharmacological Implications

: Several studies have shown that high plasma concentrations of asymmetric dimethylarginine (ADMA), a known endogenous competitive inhibitor of endothelial nitric oxide synthase (eNOS), correlate with the severity of coronary artery disease (CAD), with worsening of cardiac ischemia/reperfusion (I/R) injury and coronary atherosclerosis. It is believed that it may be an important risk factor for myocardial infarction. ADMA, when in high concentrations, can determine a significant decrease in the synthesis and bioavailability of NO (Nitric oxide) and therefore alter the mechanisms of regulation of coronary vasodilation and vasomotor function of epicardial coronary arteries. Higher serum ADMA concentration is associated with worsening of post-ischemic remodeling since coronary angiogenesis, vasculogenesis, and collateral coronary growth are seriously impaired. In addition, there are reasons to believe that elevated plasma ADMA levels are related to the development of diseases affecting coronary microcirculation, such as ischemic non-obstructive coronary artery disease (INOCA). With the aim of providing the pharmacologist engaged in the design and discovery of new ADMA-lowering drugs with a complete examination of the subject, in this review, we discuss the most important studies related to the correlations between serum ADMA levels and cardiovascular diseases mentioned above. In addition, we critically discuss the main aspects of enzymology, synthesis, and metabolism of ADMA as a prerequisite for understanding the molecular mechanisms through which high concentrations of ADMA could contribute to promoting cardiovascular diseases. ADMA represents a new target for pharmacological modulation of cardiovascular endothelial function and therefore, there is a possibility of using selective pharmacological ADMA lowering drugs in cardiovascular disease with endothelial dysfunction and high plasma ADMA levels.

Abstract We018: The Molecular Mechanism of Coronary Collateral Growth Induced by Repetitive Ischemia by Single Cell-RNA Sequencing

Coronary heart diseases (CHD) are the leading cause of mortality and morbidity in the United States. A well-developed coronary collateral circulation ameliorates the consequences of CHD, reducing the incidence of sudden death and infarct sizes following coronary occlusion. Stimulation of coronary collateral growth (CCG) is also an alternative therapeutic approach for patients with intractable angina pectoris. The importance of CCG is undisputable, but the process and mechanism underlying the CCG is unclear. We developed a mouse model of CCG by repetitive ischemia (RI) in adults, and the CCG was validated with contrast echo to measure the coronary blood flow 3D images of micro-CT to quantitate the CCG. In this study, we use single cell-RNA sequencing (scRNA-Seq) to analyze the molecular signaling of CCG at different time points during the RI process. We digested the hearts from RI days 5, 10, and 17 and control (naïve) mice, harvested non-cardiomyocytes, performed scRNA-Seq with 10x Genomics and sequenced with Next-Seq 500 (Illumina).The fastq files are converted to a gene count matrix using kallisto|bustools with refdata-gex-mm10-2020-A employed as the reference mouse genome. The Kallisto bustools output is then converted in R to files compatible for uploading to Cellenics, an open-source platform for single-cell RNA-Seq analysis. Dimensionality reduction is done with Principal Components Analysis (PCA) with 30 principal components. We will use UMAP to visualize cell clusters. Cellenics uses scType to annotate the cell populations automatically when building clusters of cell populations. Our preliminary data show dynamic kinetics of non-cardiomyocytes induced by RI over the timeline of CCG and different cell populations of non-cardiomyocytes and EC expression profiling varied in response to RI throughout CCG. We will validate the critical signaling in CCG and explore the potential targets to induce CCG.

Transient formation of collaterals contributes to the restoration of the arterial tree during cardiac regeneration in neonatal mice

Revascularization of ischemic myocardium following cardiac damage is an important step in cardiac regeneration. However, the mechanism of arteriogenesis has not been well described during cardiac regeneration. Here we investigated coronary artery remodeling and collateral growth during cardiac regeneration. Neonatal MI was induced by ligature of the left descending artery (LAD) in postnatal day (P) 1 or P7 pups from the Cx40-GFP mouse line and the arterial tree was reconstructed in 3D from images of cleared hearts collected at 1, 2, 4, 7 and 14 days after infarction. We show a rapid remodeling of the left coronary arterial tree induced by neonatal MI and the formation of numerous collateral arteries, which are transient in regenerating hearts after MI at P1 and persistent in non-regenerating hearts after MI at P7. This difference is accompanied by restoration of a perfused or a non-perfused LAD following MI at P1 or P7 respectively. Interestingly, collaterals ameliorate cardiac perfusion and drive LAD repair, and lineage tracing analysis demonstrates that the restoration of the LAD occurs by remodeling of pre-existing arterial cells independently of whether they originate in large arteries or arterioles. These results demonstrate that the restoration of the LAD artery during cardiac regeneration occurs by pruning as the rapidly forming collaterals that support perfusion of the disconnected lower LAD subsequently disappear on restoration of a unique LAD. These results highlight a rapid phase of arterial remodeling that plays an important role in vascular repair during cardiac regeneration.

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.

Abstract P1020: Differential Contribution Of Bone Marrow Cells To The Heart In Steady-state And After Repetitive Ischemia

Multiple studies have implicated the important role of migration of bone marrow (BM)-derived cells in heart after ischemia. Various BM-derived cells have been demonstrated to exert beneficial or detrimental effects in ischemia. But the response of BM to repetitive ischemia (RI) and the difference in contribution of this heterogenous population to the heart after RI remain elusive. Here, we applied an occluder on the left anterior descending coronary arteries of rats and they went under RI over a period of 10-17 days. This RI model induced coronary collateral growth and increased myocardial blood flow in the ischemic region after 10-17 days. We performed single- cell RNA sequencing and analysis of 24103 bone marrow transcriptomes isolated from the heart and bone marrow of rats in steady-state and after RI. Unsupervised clustering of cardiac neutrophils revealed 28 major clusters and 16 different cell types. Compared to steady state heart, proportion of BM derived macrophage mainly CD163 positive population was increased in the heart after RI. The proportion of BM derived natural killer cells was decreased markedly in the RI group. There was a slightly higher percentage of BM derived endothelial cells and lower smooth muscle cell and fibroblast in the RI group. While neutrophil degranulation, leukocyte activation, leukocyte migration are the major biological pathways in both groups, RI group showed activation of regeneration. We report heterogeneity of BM cells migrated to the heart during RI and redefine the BM that respond to heart ischemia and participate in induction of angiogenesis after RI.

Implication of collateral growth in the process of cardiac regeneration after neonatal infarction in mice

The recent discovery of the capacity of the mammalian heart to completely regenerate during a short period of time after birth opened a new area of research in the cardiovascular field. The revascularization of the ischemic area is an important process to favor cardiac regeneration and we have previously shown that preexisting coronary arteries undergo major remodeling events following adult MI. This mechanism of arteriogenesis is known as collateral growth but it has not been well described during cardiac regeneration. We hypothesize that collateral growth occurs more frequently in regenerative hearts compared to non-regenerative ones. To investigate the coronary arteries remodeling and collateral growth during cardiac regeneration after neonatal myocardial infarction (MI). Neonatal MI was performed by the permanent ligation of the left coronary artery on one day-old (P1) and seven day-old (P7) mice. MI surgery was performed on P1 or P7 pups from the Cx40-GFP mouse line in which the fluorescent protein GFP is specifically expressed in coronary arteries. Hearts were collected at 1, 2, 4, 7 and 14 dpi and processed for clearing using CUBIC solution. 3D reconstruction was acquired from images of cleared hearts. Our data show the dynamic remodeling of the ligated coronary artery, which completely regenerates in two weeks, starting one day-post-injury (dpi) by the migration of isolated arterial endothelial cells migrating from the injured artery to form a new vessel at 4dpi. We observed a larger number of collaterals branching from the injured left artery to healthy right or septal arteries in regenerating hearts (P1) in comparison with non-regenerating hearts (P7). These results highlight the rapid phase of arterial remodeling that would play an important role in inducing cardiac regeneration.

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 is incompletely 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 (RI) and investigate the process of coronary collateral growth in adult hearts by lineage tracing.Methods and Results– A murine model of coronary collateral growth was developed using short episodes of repetitive ischemia. Repetitive ischemia resulted in robust collateral growth in adult mouse hearts, validated by high‐resolution micro‐computed tomography. ROSA mT/mG reporter mice with Apln‐CreERT2 transgene were used for lineage tracing of sprouting endothelial cells to determine cellular contributions to coronary collateral growth. Two‐photon and confocal microscopy imaging revealed RI‐induced coronary collateral growth initiated from sprouting Apelin+GFP+endothelial cells. Newly formed capillaries in the collateral‐dependent zone expanded in diameter upon RI stimulation and arterialized with smooth muscle cell recruitment to form mature coronary collateral arteries. Notably, pre‐existing coronary arteries and arterioles were not Apelin+ GFP+, and all Apelin+endothelial cells arose from sprouting capillaries. HIF1α, VEGFR2, CXCR4, MCP‐1, and JAG1 signaling in the collateral‐dependent zone were also upregulated during coronary collateral growth.Conclusions– We developed a murine model of RI‐induced coronary collateral growth, and our data show sprouting EC contribute to coronary collateral growth in adult hearts. Sprouting angiogenesis is shown for the first time to arterialize into mature blood vessels of larger diameter in response to repetitive ischemia in adult hearts.

The essential role for endothelial cell sprouting in coronary collateral growth

RationaleCoronary 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. ObjectiveTo 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 resultsA 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. ConclusionsWe 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.

The role of CXCR4 in vascular endothelial cells in myocardial ischemia

CXCR4 and CXCL12 signaling pathway is important for stem cell homing in myocardial ischemia and cardiovascular regeneration. Recently report shows that CXCR4 and CXCL12 signaling pathway is involved in coronary collateral growth in myocardial ischemia. Endothelial cells are a key players in the coronary collateral growth. In this study, we use genetically modified mice CXCR4 endothelial cell specific knockout mice to study the role of CXCR4 in endothelial cells in myocardial ischemia. First, we study the role of CXCR4 in endothelial cells in coronary collateral growth in our mouse model by inducing repetitive ischemia. Second, we use the mouse myocardial infarction model to study the role of CXCR4 in endothelial cells in cardiovascular regeneration. We measure the cardiac function, vessel density and coronary blood flow in the the heart. Our preliminary data showed that the deletion of CXCR4 changes cardiac function and coronary collateral growth and the CXCR4 and CXCL12 signaling pathway is essential to cardiovascular regeneration. It suggested that CXCR4/CXCL12 signaling pathway as a potential target for the development of therapeutic treatment for coronary artery diseases and prevention of heart attack.

The role of Gdf11 in Coronary Collateral Growth

Cardiovascular disease is the leading cause of death in the United States of America, being responsible for 610,000 deaths annually. One in seven of which are related to Ischemic Heart Disease (IHD). Collateral growth is found to be vital to cardiovascular health, with those who have poor collateral networks having a more difficult time recovering and are more likely to have a recurring problem than those with healthy collateral networks. In this study, we investigate if GDF11 affects these collateral networks. To further investigate this, we will use the GDF11 deficient mice and wild type control mice. GDF11 is controversial in its function in aging. However, our study is more focused on the function of GDF11 in coronary collateral growth. We will compare the coronary collaterals among the following groups: 1) naïve wild type and Gdf 11 knockout mice without any intervention; 2) wild type and Gdf 11 knockout mice whose left anterior descending artery (LAD) had been ligated, to mimic a myocardial infarction; 3) wild type and Gdf 11 knockout mice with repetitive ischemia stimulation. Microfil and micro-CT will be used to measure the coronary collaterals and contrast echocardiograph will be used to measure the coronary blood flow. These structure and function data will demonstrate the role of GDF 11 in coronary collateral growth.

Implication of collateral growth in the process of cardiac regeneration after neonatal myocardium infarction in mice

The recent discovery of the capacity of the mammalian heart to completely regenerate during a short period of time after birth opened a new area of research in the cardiovascular field. The revascularization of the ischemic area is an important process to favor cardiac regeneration and we have previously shown that pre-existing coronary arteries undergo major remodeling events following adult MI. This mechanism of arteriogenesis is known as collateral growth but it has not been well described during cardiac regeneration. We hypothesize that collateral growth occurs more frequently in regenerative hearts compared to non-regenerative ones. To investigate the coronary arteries remodeling and collateral growth during cardiac regeneration after neonatal myocardial infarction (MI). Neonatal MI was performed by the permanent ligation of the left coronary artery on one day-old (P1) and seven day-old (P7) mice. MI surgery was performed on P1 or P7 pups from the Cx40-GFP mouse line in which the fluorescent protein GFP is specifically expressed in coronary arteries. Hearts were collected at 1, 2, 4, 7 and 14 dpi and processed for clearing using CUBIC solution. 3D reconstruction was acquired from images of cleared hearts. Our data show the dynamic remodeling of the ligated coronary artery, which completely regenerates in two weeks, starting one day-post-injury (dpi) by the migration of isolated arterial endothelial cells migrating from the injured artery to form a new vessel at 4dpi. We observed a larger number of collaterals branching from the injured left artery to healthy right or septal arteries in regenerating hearts (P1) in comparison with non-regenerating hearts (P7). These results highlight the rapid phase of arterial remodeling that would play an important role in inducing cardiac regeneration.

Sprouting Angiogenesis Contributes to Coronary Collateral Growth Induced by Repetitive Ischemia in Adult Mice

In the United States, coronary heart diseases (CHD) are the leading cause of mortality and morbidity. A well‐developed coronary collateral circulation ameliorates the consequences of CHD, reducing the incidence of sudden death and infarct sizes following coronary occlusion. Stimulation of coronary collateral growth (CCG) is also an alternative therapeutic approach to patients with intractable angina pectoris. The importance of CCG is undisputable, but the process and mechanism underlying CCG is unclear. We developed a mouse model of CCG induced by repetitive ischemia (RI) and validated CCG by contrast echocardiography to measure the coronary blood flow in the normal zone (NZ) and the collateral dependent zone (CZ). We also used Micro‐CT scans to reconstruct the coronary vasculature in 3D and quantify CCG. In this study, we crossed ROSA mT/mG double fluorescent reporter mice with cell type‐specific cre mice to investigate the roles that different cell‐types play at various stages of CCG. Mice were subjected to the RI protocol and their cardiac function and coronary blood flow was measured by echocardiography. Following sacrifice, hearts were fixed for immunostaining or imaging under confocal and multiphoton microscopy. In ROSA mT/mG floxed, Apln cre mouse hearts, large vessels (diameter of 20–50 μm) expressing GFP were identified in the collateral dependent zone in the early stage of RI, indicating that they originated from sprouting endothelial cells. At the end of RI, a complete network of these vessels was observed anastomosing with existing vasculature from the NZ. Immunostaining revealed an outer layer of smooth muscle cells in these vessels, suggesting the arteriogenesis or arterialization. Interestingly, in ROSA mT/mG floxed, Lyz2 cre mouse hearts, GFP expressing myeloid cells were observed in high numbers around newly formed large vessels, suggesting that immune cell recruitment occurs in the early stages of CCG. Our preliminary data show that coronary collaterals form in response to RI, are of microvascular origin and are associated with early recruitment of immune cells to the CZ. Our further study will focus on the process of CCG in greater detail at the molecular and cellular level, potentially leading toward a therapeutic application of induced CCG in patients of ischemic heart diseases.Support or Funding InformationThe research is funded by National Institutes of Health grant 2R01HL103227‐05 (YZ, LY), 1R01HL135110‐01 (WMC, LY), 1R01 HL137008‐01A1 (LY) from American Heart Association (LY).

Regulation of Coronary Collateral Growth by MicroRNA‐21 in Metabolic Syndrome

Well‐developed coronary collaterals prove to be highly beneficial in salvaging ischemic myocardium, preserving cardiac function, and improving patient outcome post‐occlusion. However, this process of coronary collateral growth (CCG) is impaired in patients with metabolic syndrome. A complete understanding of the underlying mechanism, cell types, and genes contributing towards this impairment have yet to be elucidated. Therefore, uncovering more about the process may lead to potential therapeutics to induce CCG in metabolic syndrome. MicroRNA‐21 (miR‐21) is abundantly expressed in vascular and immune cells with numerous implications in cardiovascular disease including atherosclerosis, heart failure, and myocardial infarction. Furthermore, miR‐21 has been shown to regulate processes such as apoptosis, immune cell polarization, and endothelial progenitor proliferation. Additionally, miR‐21 dysregulation has been rooted in the diabetic population where lies a systemic inflammatory state. In this study, we investigated the role of miR‐21 in a mouse model of CCG.Our preliminary data suggested that down‐regulating miR‐21 rescues impaired CCG in a diet‐induced model of metabolic syndrome. Thus, we investigated the underlying mechanism and focused on the roles of miR‐21 in the maintenance of vascular homeostasis, function and inflammatory responses in metabolic syndrome. First, we studied whether miR‐21 regulates endothelial homeostasis by modulating the function and homing of bone marrow stem cells in metabolic syndrome. We analyzed endothelial progenitor cells (CD34+) in bone marrow and peripheral blood, along with endothelial proliferation in WT, miR‐21 knockout, and metabolic syndrome mice. Second, we studied whether miR‐21 regulates the inflammatory response in metabolic syndrome by characterizing bone marrow derived macrophages from WT and miR‐21 knockout animals along with mapping their influence on vascular cells. Third, we utilized a myeloid specific miR‐21 knockout to study its role during CCG in vivo. We found CCG to be blunted in these animals which suggests the importance of myeloid derived miR‐21 in healthy mice. Further studies will give us more insight on miR‐21 and its regulation of CCG in metabolic syndrome.Support or Funding InformationThe research is funded by National Institutes of Health grant 2R01HL103227‐05 (YZ, LY), 1R01HL135110‐01 (WMC, LY), 1R01 HL137008‐01A1 (LY)from American Heart Association (LY).

Abstract 592: Cardioprotection During Ischemia by Induced Coronary Collateral Growth

Patients with poorly developed coronary collateral networks have a poorer prognosis after a cardiovascular event than those with well-developed collaterals. The outcome of patients with metabolic syndrome (MetS) was worse than the patient without MetS. Interestingly, coronary collateral growth is impaired in MetS. We hypothesize that coronary collaterals are critical for the cardiac protection in ischemic heart diseases (IHD) and induction of coronary collateral growth (CCG) might ameliorate the outcome of patients with MetS. We study the cardiac protection by coronary collaterals in IHD and the underlying mechanism of impaired CCG in the MetSin a mouse model of CCG. A pneumatic snare was implanted and situated around the LAD. After the mice recovery from the surgery, periodic inflation of the snare occludes the LAD; thus, producing repetitive ischemia (RI). Cardiac function was measure with echocardiography. CCG was measured by myocardial blood flow with contrast echocardiography and by collateral numbers with micro-CT or by vascular density with fluorescence scope. Our preliminary results showed that with occlusion of the LAD, there was no blood flow in the ischemic area. However, after stimulation of CCG by RI, the blood flow the ischemic area was compensated by the grown coronary collaterals and cardiac function was reserved during ischemia. Diabetic mice failed to grow coronary collaterals after RI stimulation and there was no cardiac protection from coronary collaterals during ischemia. Moreover, we studied the roles of growth differentiation factor 11 (GDF11) and miR21 in the regulation of CCG and cardiac protection in IHD. GDF11 was downregulated and miR21 was upregulated in the hearts of Zucker Obese Fatty (ZOF) rats during CCG. We used the Gdf 11 knockout mice and miR 21 knockout mice to study the underlying mechanism. GDF11 is highly expressed in myocardial blood vessels, which suggested the role of GDF11 in vascular growth. Impaired CCG of mice on high fat and high sugar was restored by miR-21 knockout. These data suggest that miR-21 is involved in the impairment of CCG in MetS.

Myeloid Derived miR‐21 Influences Coronary Collateral Growth in Metabolic Syndrome

Well‐developed coronary collaterals prove to be highly beneficial in salvaging ischemic myocardium, preserving cardiac function, and improving patient outcome post‐occlusion. However, this process is impaired in patients with metabolic syndrome. A more complete understanding of the underlying mechanism, cell types, and genes causing the impaired coronary collateral growth (CCG) response have yet to be elucidated. Better understanding of the process of CCG will lead to potential therapeutics to induce CCG and circumvent ischemic injury. MicroRNA‐21 (miR‐21) has been studied in cancer, with its central role in the down‐regulation of pro‐apoptotic genes leading to sustained cellular proliferation. However, miR‐21 has recently been reported to modulate inflammation after myocardial infarction (MI) and to play a role in the development of atherosclerosis. Furthermore, miR‐21 has been shown to be involved in macrophage polarization, where it generally serves to reduce inflammatory markers and promote anti‐inflammatory markers. In addition, miR‐21 dysregulation has been rooted in the diabetic population wherein lies a systemic inflammatory state. In this study, we investigated the role of miR‐21 during CCG in diabetes mellitus, which is a major risk factor for the development of cardiovascular disease. Our preliminary data suggested that down‐regulating miR‐21 rescues the impaired CCG in diet‐induced diabetic animals. Given that miR‐21 is highly abundant in immune cells, we sought to further study if miR‐21 regulates CCG by modulating the inflammatory response. While macrophages have been extensively studied during MI events or in atherosclerosis conditions, their roles during CCG are not well‐defined. Thus, we utilize a myeloid specific (Lyz2) miR‐21 knockout to investigate the impact of miR‐21 in macrophages on CCG within the context of diabetes. First, colony stimulating factor‐1 directed bone marrow derived macrophages from miR‐21 Lyz2‐knockout mice were subjected to high glucose (4.5 g/L) and / or hypoxia (3% oxygen) to mimic diabetic conditions. Interestingly, the knockout macrophages have differences in their spindle like projections even before beginning treatment. This possibly suggests greater baseline activation compared to the control. We also observe morphological differences after treatment. The expression profile will give more accurate results. In vivo, CCG is blunted in the miR‐21 Lyz2‐knockout animals subjected to the repetitive ischemia protocol. This suggests that miR‐21 in the myeloid lineage is critical for CCG in healthy non‐diabetic mice. Further studies of CCG in the miR‐21 Lyz2‐knockout mice fed high fat and high sugar diet will give us more insight into the mechanisms underlying impaired CCG in diabetes.Support or Funding InformationThe research is funded by National Institutes of Health grant 2R01HL103227‐05 (Zhang, Yin), 1R01HL135110‐01 (WMC, LY), 1 R01 HL137008‐01A1 (LY), 1R15HL115540‐01 (LY) and 14BGIA18770028 from American Heart Association.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Correlative, Three‐dimensional Approach to Studying Coronary Collateral Growth Using Lineage Tracing, Micro‐computed Tomography and Multiphoton Imaging in a Mouse Model of Repetitive Ischemia

Coronary heart diseases (CHD) are the leading cause of mortality and morbidity in the United States. Inherent coronary collateral network in the heart represents a native circulation that is capable of undergoing tremendous abluminal expansion to provide flow to an ischemic area of the myocardium. A well‐developed coronary collateral circulation ameliorates the consequences of CHD, reducing the incidence of sudden death and infarct size following coronary occlusion. Stimulation of coronary collateral growth (CCG) is an alternative therapeutic approach for patients with intractable angina pectoris who are not indicated for percutaneous coronary intervention and/or coronary artery bypass grafting. Even though the importance of CCG is undisputable, the mechanism underlying the CCG is not clear, such as its time course and sequence of process, critical cell type(s) and genes/factors. We developed a mouse model of CCG by repetitive ischemia (RI) and validated the CCG with contrast echo to measure the coronary blood flow and 3D imaging using micro‐CT (μCT) to quantitate the CCG. Figure 1B shows the micro‐CT images of grown coronary collaterals after RI while Figure 1A shows no native coronary collaterals in mouse heart. Moreover, we adopted lineage tracing to dissect the process of CCG. We crossed lineage tracing (ROSA mT/mG floxed) mice with tissue specific cre expressing lines (endothelial cells, smooth muscle cells, inflammatory cells, etc.) and these mice were subjected to RI surgery and protocol. Mice were echoed for blood flow measurement and sacrificed at various time‐points. Mouse hearts were harvested for micro‐CT or multi‐photon fluorescent imaging. Tiled and stitched z‐stacks of heart slices containing the growing collaterals were acquired for fluorescent target cells as well as vasculature. Figure 2 shows vasculature of a ROSA‐tie2‐GFP mouse heart by multiphoton without RI. Also, datasets from μCT and multiphoton modalities could be merged in Amira™ so that the final 3D reconstruction included precise locations of lineage cells within the growing collaterals. Post‐imaging, the heart slices could also be embedded and sectioned for fluorescent IHC for proliferation and other proteins critical for arteriogenesis. In conclusion, we have developed a method for exploring the process of CCG in greater detail at the molecular and cellular level in a mouse CCG model. Understanding this process at such level could provide a therapeutic strategy to induce CCG in ischemic heart diseases.Support or Funding InformationThe research is funded by National Institutes of Health grant 2R01HL103227‐05 (YZ, LY), 1R01HL135110‐01 (WMC, LY), 1R01 HL137008‐01A1 (LY), 1R15HL115540‐01 (LY) and 14BGIA18770028 from American Heart Association (LY).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

P1734Physical exercise induces coronary artery collateral growth and affects cardiovascular biomarkers: A biomarker sub-analysis of the EXCITE trial

P1734Physical exercise induces coronary artery collateral growth and affects cardiovascular biomarkers: A biomarker sub-analysis of the EXCITE trial

Impaired Coronary Collateral Growth in a Mouse Model of Diabetes

Cardiovascular disease is the leading cause of death in the United States, Ischemic Heart Disease (IHD) accounts for one in seven cardiovascular related deaths. Patients with poorly developed coronary collateral networks have a poorer prognosis after a cardiovascular event than those with well‐developed collaterals. Furthermore, coronary collateral growth is impaired in metabolic syndromes such as diabetes, and obesity. The underlying mechanism(s) that is (are) causal for growth are unknown. In an attempt to elucidate a mechanistic understanding, we developed a murine model that allows us to use genetically modified mice to study the coronary collateral growth spatially and temporally.Methodsa pneumatic snare was implanted and situated around the LAD (open chest surgery during anesthesia). After mice recovered from surgery, the snare was periodically inflated to occlude the LAD; thus producing episodes of ischemia or repetitive ischemia (RI). Coronary collateral growth (CCG) was measured by myocardial blood flow with contrast echocardiography and by collateral numbers by micro‐CT.Results1) No native coronary collaterals were found in mice. 2) After the RI stimulation, extensive coronary collaterals grew. 3) Diabetic mice failed to grow coronary collaterals after RI stimulation. 4) Moreover, the coronary blood flow increased in the ischemic area when there was coronary collateral growth. 5) The quantification of vessel density by tree analysis showed an increase after RI stimulation. Figure 1 (A–C) shows the microfil pictures of the wild type mouse heart without CCG (A), the wild type mouse heart with CCG (B), and the diabetic heart without CCG (C). We believe this murine model of coronary collateral growth is very useful for future studies to investigate the mechanism of regulation of coronary collateral growth. Figure 1 (D–F) shows the micro‐CT pictures of the wild type mouse heart without CCG (D), the wild type mouse heart with CCG (E) and the diabetic heart without CCG (F).Support or Funding Information2R01HL103227‐05( Zhang, Yin), 1R01HL135110‐01 (WMC, LY), 1 R01 HL137008‐01A1 (LY), 1R15HL115540‐01 (LY) and14BGIA18770028 from American Heart AssociationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of MicroRNA‐21 Knockout on Coronary Collateral Growth in Metabolic Syndrome

A highly‐functional coronary collateral circulation proves beneficial during acute coronary occlusions such as myocardial infarctions, but the molecular drivers of coronary collateral growth (CCG) have yet to be completely understood. Our previous work shows that CCG is impaired in Zucker Obese Fatty (ZOF) rats (a rat model of metabolic syndrome). These results are consistent with what is seen in human patients with metabolic syndrome. Our gene expression profiling of rat hearts during repetitive ischemia (RI) in ZOF rats and Zucker lean (ZLN) rats showed microRNA‐21 (miR‐21) to be one of the most upregulated genes, and was found to be significantly increased in ZOF rat hearts compared to ZLN rat hearts. This expression was also validated by quantitative RT‐PCR. Interestingly, miR‐21 was reported to play a critical role during CCG in a JCR rats (another rat model of metabolic syndrome) where treatment with a miR‐21 antagomir improved RI‐induced CCG. Since miR‐21 antagomirs may target any metabolically active tissue such as liver, intestine and heart, it is unknown how the knockdown of miR‐21 ameliorate CCG. The miR‐21 knockout (KO) mouse will help us to investigate this question. First, we mapped out where miR‐21 expression occurs by LacZ staining in a miR‐21 mice with a LacZ gene knockin. Staining has shown that there is an abundance of miR‐21 expressed in the heart, aorta, and muscle vasculature of mice. We then investigated the impact that genetic ablation of miR‐21 has on coronary collateral formation in a mouse model of metabolic syndrome induced by a high fat high sugar (HFHS) diet. A pneumatic balloon is surgically placed over the left anterior descending artery (LAD), and after a recovery period the mice underwent RI protocol. CCG was measured by myocardial blood flow with contrast echocardiography and by collateral numbers using micro‐computed tomography (microCT). Interestingly, the right coronary artery (RCA) of miR‐21 KO mice were poorly perfused, possibly suggesting miR‐21 is a player in coronary artery development or potentially in the development of atherosclerotic lesions. Impaired CCG of mice on HFHS diet was restored by miR‐21 KO. Moreover, miR‐21 expression levels were increased in induced vascular progenitor and endothelial cells subjected to high fat and high glucose treatment. miR‐21 expression was also increased in diabetic (db/db) and obese (ob/ob) mice. These data suggest that miR‐21 is a key player in the cardiovascular system in metabolic syndrome and is likely to be involved in the impairment of CCG in metabolic syndrome.Support or Funding InformationThe research is funded by National Institutes of Health grant 2R01HL103227‐05( Zhang, Yin), 1R01HL135110‐01 (WMC, LY), 1 R01 HL137008‐01A1 (LY), 1R15HL115540‐01 (LY) and 14BGIA18770028 from American Heart Association.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.