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.

Amplification of coronary arteriogenic capacity of multipotent stromal cells by epidermal growth factor.

OBJECTIVE: We determined whether increasing adherence of multipotent stromal cells (MSCs) would amplify their effects on coronary collateral growth (CCG). METHODS AND RESULTS: Adhesion was established in cultured coronary endothelials cells (CECs) or MSCs treated with epidermal growth factor (EGF). EGF increased MSCs adhesion to CECs, and increased intercellular adhesion molecule (ICAM-1) or vascular cell adhesion molecule (VCAM-1) expression. Increased adherence was blocked by EGF receptor antagonism or antibodies to the adhesion molecules. To determine whether adherent MSCs, treated with EGF, would augment CCG, repetitive episodes of myocardial ischemia (RI) were introduced and CCG was measured from the ratio of collateral-dependent (CZ) and normal zone (NZ) flows. CZ/NZ was increased by MSCs without treatment versus RI-control and was further increased by EGF-treated MSCs. EGF-treated MSCs significantly improved myocardial function versus RI or RI+MSCs demonstrating that the increase in collateral flow was functionally significant. Engraftment of MSCs into myocardium was also increased by EGF treatment. CONCLUSIONS: These results reveal the importance of EGF in MSCs adhesion to endothelium and suggest that MSCs may be effective therapies for the stimulation of coronary collateral growth when interventions are used to increase their adhesion and homing (in vitro EGF treatment) to the jeopardized myocardium.