The essential role for endothelial cell sprouting in coronary collateral growth

Abstract

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.

Keywords: Arterialization; Coronary circulation; Coronary collateral growth; Ischemic heart diseases; Postnatal coronary collateral formation; Repetitive ischemia.

Figure 1.

Mouse model of coronary collateral growth by repetitive ischemia (RI). (A) Timeline of the RI protocol. (B) Schematic showing surgical placement of pneumatic snare around left anterior descending (LAD) artery for RI protocol. Normal zone (NZ) denotes the area above the pneumatic snare and collateral-dependent zone (CZ), the area below the pneumatic snare (ischemic during snare inflation). During the occlusion of LAD, CZ blood flow is dependent on the degree of coronary collaterals. (C) Brightfield (BF) image of a Microfil® perfused heart showing pneumatic snare placement over the LAD artery. (D-E) BF image of a Microfil® perfused mouse hearts without (D) and with (E) RI. (F-G) Micro-computed tomographic (micro-CT) reconstruction of hearts without (F) and with (G) RI. D and F represent filling of the coronary arterial circulation upon acute LAD artery ligation. Note the large area (white oval) without filling, indicating a lack of collaterals or vessels too small to be visualized. E and G show extensive collateral growth (white oval) in a mouse heart subjected to RI. (H) Magnification of G. In F-H, the white filling represents the right coronary artery (RCA) perfusion territory, green is the septal artery, blue is the LAD, and yellow and purple vessels denote likely and definite collaterals, respectively.

Figure 2.

Coronary vascular architecture and vascular tree analysis of micro-computed tomographic reconstruction images of Microfil® perfused hearts. (A) Vascular tree from a naïve mouse heart. (B) Vascular tree from a mouse heart with RI. (C) Total arterial branches and branching ends in the heart from A. (D) Total arterial branches and branching ends in heart from B. Yellow (aortic root), red (end branches), blue (parent branches). (E) The numbers of total branches and branch ends of coronary vascular tree in mice with coronary collateral growth (CCG) and without CCG (n=3 mice per group, two-way AN OVA (Sidak’s multiple comparison test) was used for statistical analysis, *P <0.05).

Figure 3.

Coronary vascular architecture and vascular tree analysis of micro-computed tomographic reconstruction images of main branches of Microfil® perfused hearts. (A) Vascular trees from a mouse without coronary collateral growth (CCG). (B) Vascular trees from a mouse with CCG. Maps of arterial branches and branching ends for trees from the left coronary artery (LCA), the right coronary artery (RCA) and septum are shown, respectively. Yellow (aortic root), red (end branches), blue (parent branches). (C) The numbers of branches and branch ends in LCA, RCA and Septum of A and B.

Figure 4.

Two-photon microscopic images of native heart slices (1mm) from pan-endothelial (Tie2-cre⁺) and actively sprouting endothelial (Apln-cre⁺) mT/mG mice. (A) Native Tie2-cre⁺ heart with cells expressing Tie2 (GFP⁺). Regions of interests (ROIs; pink boxes) in A are shown in greater detail in B and E, where some Tie2⁻cells are also Td-tomato⁺. (C) Native Apln⁺ heart with cells expressing Apelin (GFP⁺). ROIs in C are shown in greater detail in D and F, where apelin⁻ cells are Td-tomato⁺. Apln⁺ GFP⁺ cells represent endothelial sprouting in the native heart.

Figure 5.

Sprouting and proliferating endothelial cells (ECs) in the mouse heart during early stages of coronary collateral growth (CCG; induced by 5 days of repetitive ischemia [RI]). Images of normal zone (NZ) (A) and collateral dependent zone (CZ) (B) of mT/mG x Apln-cre mice after 5-day RI, showing more sprouting ECs in the CZ than ECs in NZ. Apln⁺ cells are GFP⁺. (C) 5-ethynyl-2’-deoxyuridine (EdU) labeling scheme of proliferating cells. EdU staining in the NZ (D) and CZ (E) of mT/mG x Apln-cre heart after 2-day’s EdU injections. Apln⁺ cells are labeled with anti-GFP antibody. (F) High magnification of EdU⁺ staining images in control (con) apex and RI CZ with red arrows marking co-localization of EdU⁺ and GFP⁺. (G-H) Quantitation of EdU⁺ nuclei (proliferating cells) and EdU⁺Apln⁺ (sprouting and proliferating EC) shows hearts with RI have more EdU⁺ or EdU⁺Apln⁺ cells (n= 12 fields, 4 fields per animals, unpaired t-test with Welch’ correction was used for statistical analysis, *P<0.05)

Figure 6.

Proliferating endothelial cells (ECs) in the heart during late stages of coronary collateral growth (CCG; induced by 8-10 days of repetitive ischemia [RI]). (A) Labeling scheme of proliferating cells with 5-ethynyl-2’-deoxyuridine (EdU) and 5-bromo-2’-deoxyuridine (BrdU). Images of normal zone (NZ) (B) and collateral dependent zone (CZ) (C) of mT/mG x Apln-cre mouse hearts after 17-day RI, showing more proliferating ECs in the CZ than NZ. Apln⁺ cells are labeled with anti-GFP antibody. (D and E) High magnification of EdU/BrdU staining in NZ (D) and CZ (E). (F) Quantitation of Apln⁺EdU⁺ nuclei (GFP⁺ proliferating ECs at the early stage of RI) and Apln⁺BrdU⁺ nuclei (GFP⁺ proliferating ECs at the late stage of RI) in NZ and CZ (n= 12 fields, 4 fields per animals, two-way ANOVA (Sidak’s multiple comparison test) was used for statistical analysis, *P <0.05).

Figure 7.

Newly formed capillary expansion in response to repetitive ischemia (RI). (A-B) Images of the normal zone (NZ) and collateral dependent zone (CZ) from mT/mG x Apln-cre sham heart. (C-D) Images of the NZ and CZ from mT/mG x Apln-cre heart after 17-day RI. Capillary expansion occurs more in the CZ than NZ of RI heart in response to RI or more in the NZ or the CZ of the sham heart. GFP⁺ vessels in the CZ from the RI heart are more abundant, of larger diameter and more prolonged than in the NZ. An extensive network of these remodeled capillaries can be seen in E and newly formed, and expanded capillaries can be seen up to a depth of 1000 μM from epicardial surface. Approximate diameters of these large vessels (up to 30 μM) are shown.

Figure 8.

Arterialization of newly formed blood vessels. (A-D) Images of mT/mG x Apln-cre mouse heart with 17-day repetitive ischemia (RI). (B) Tortuous, newly-formed blood vessels with GFP⁺ endothelial layer and tdTomato⁺ outer layer in the collateral-dependent zone (CZ), indicating vessel maturation with a layer of mural cells. (C) Single z-slices from the z-stack in B revealed that the outer layer marked with yellow arrows is continuous at every z-plane. The Smooth muscle alpha-actin (αSMA) layer encompassing the GFP⁺ vessel is present in the CZ (E) but not in the normal zone (NZ) (D). Single z-slices (F) from the z-stack in E showed that the α-SMA layer is present outside the endothelial monolayer at every z-plane marked with yellow arrows, suggesting the maturation of sprouting capillaries with smooth muscle cell recruitment.

Figure 9.

Hypoxyprobe® staining of wild-type (WT) mouse cardiac tissue. (A-B) Base and apex of non-surgical control heart. (C) High magnification of B. (D-E) Normal zone (NZ) and collateral-dependent zone (CZ) from 3-day repetitive ischemia (RI) hearts (F) High magnification of E. (G) The quantitation of green fluorescence of hypoxia probe in the control hearts and RI hearts. 4’,6-diamidino-2-phenylindole (DAPI) (blue), Hypoxyprobe® (green), Wheat Germ Agglutinin; WGA (red) (n= 12 fields in control and n=16 fields in RI group, 4 fields per animal, unpaired Mann-Whitney test was used for statistical analysis, *P <0.05).

Figure 10.

mRNA expression in mouse hearts at early stages (3 days) of repetitive ischemia (RI) by qPCR. (A) Compared to non-surgical controls, C-X-C motif chemokine receptor 4 (Cxcr4), Vascular Endothelial Growth Factor Receptor 2 (Vegfr2), jagged canonical Notch ligand 1 (Jag1), expression in the mouse hearts with RI were significantly increased (**P<0.01); compared to the sham control, Cxcr4, Vegfr2, Jag1 and Hypoxia Inducible Factor subunit alpha (Hif1α) were significantly increased (n=7 mice/group, one-way ANOVA (Holm-Sidak’s multiple comparison test) was used for statistical analysis, *P < 0.05, **P < 0.01). (B) Monocyte Chemoattractant Protein-1 (Mcp-1) was upregulated in mouse hearts with RI compared to sham controls (n=9 in the control group and n=4 in the RI groups, unpaired Mann-Whitney U test was used for statistical analysis, *P < 0.05). (C) Hif1α was upregulated in the collateral-dependent zone (CZ) of RI mouse hearts compared to non-surgical controls and sham controls (n=5 in the control group, n=4 in the sham groups, and n=3 in the RI groups, one-way ANOVA (Turkey multiple comparison test) was used for statistical analysis, *P < 0.05, **P < 0.01). (D) Functional protein association networks via STRING show the signaling pathway linking genes with differences in A-C. (E-G) Functional protein association networks via STRING show signaling pathways of CXCR4, JAG1 and VEGFR2.