Cooperation between VEGF and beta3 integrin during cardiac vascular development

Abstract

In the developing myocardium, vascular endothelial growth factor (VEGF)-dependent neovascularization occurs by division of existing vessels, a process that persists for several weeks following birth. During this remodeling phase, mRNA expression of beta3 integrin in the heart decreases significantly as vessel maturation progresses. However, in male mice lacking beta3, coronary capillaries fail to mature and continue to exhibit irregular endothelial thickness, endothelial protrusions into the lumen, and expanded cytoplasmic vacuoles. Surprisingly, this phenotype was not seen in female beta3-null mice. Enhanced VEGF signaling contributes to the beta3-null phenotype, because these vessels can be normalized by inhibitors of VEGF or Flk-1. Moreover, intravenous injection of VEGF induces a similar angiogenic phenotype in hearts of adult wild-type mice. These findings show a clear vascular phenotype in the hearts of mice lacking beta3 and suggest this integrin plays a critical role in coronary vascular development and the vascular response to VEGF.

Figure 1

Postnatal angiogenesis and vascular remodeling in the heart. The cardiac coronary capillary bed undergoes significant vascular remodeling during a postnatal period of myocyte hypertrophy. (A) Immunostaining for PECAM-1/CD31 shows capillaries in cross section (top) or transverse section (bottom) for 1- and 6-week-old wild-type mice. The schematic illustrates how myocytes (blue) and capillaries (brown) are organized in the adult heart for homogeneous tissue oxygenation. Using TEM, a blinded observer examined several hundred coronary capillaries per mouse and recorded the frequency of luminal filopodia, expanded vacuoles, and/or intussusception. These characteristics of immature, angiogenic blood vessels were observed more frequently in neonatal mice (1 to 3 weeks old) of either sex (B). β3 integrin mRNA expression, considered a marker of angiogenic endothelium, decreases rapidly with age following birth on a similar time scale as capillary maturation (C). (A) Scale bar = 5 μm. (B-C) Data represent mean ± SE.

Figure 2

β3 integrin protein expression in the mouse heart. Immunostaining serial sections for PECAM-1 and β3 integrin confirms the hamster antimouse antibody can detect β3 protein expression in wild-type (but not β3-null) mouse hearts (A). During the first few postnatal weeks, β3 protein expression decreases and is only faintly detected on smooth muscle cells on small arteries in the adult heart (B). In late-stage embryo hearts, β3 integrin is found near areas of vascular remodeling (C, arrows indicate clusters of vessels) or associated with vascular structures (D, left), interstitial cells (D, middle), and myocytes (D, right). Bar = 100 μm (A-B) or 200 μm (C-D).

Figure 3

Immature coronary capillaries in the male β3-null mouse heart. Using TEM, we evaluated whether coronary capillary maturation is affected by null expression of β3 integrin. Whereas the frequency of immature vessels decreases with age in male and female wild-type mice as well as female β3-null mice, β3-null mice continue to display these immature characteristics, even in senescent (27-week-old) mice.

Figure 4

Male β3-null coronary capillaries appear immature and angiogenic.Transmission electron micrographs show that coronary capillaries from adult wild-type mice (A-B) are composed of endothelial cells with a smooth luminal surface and uniform thickness around the vessel perimeter. In contrast, coronary capillaries from adult male β3-null mice (C-E) often have irregular endothelial-cell thickness (arrows) along with filopodia extending into the vessel lumen (arrows). The luminal filopodia can originate from cell-cell junctions as well as from the central region of a single endothelial cell (E). All filopodia appear to extend toward the vessel lumen rather than into the extravascular space. The frequent presence of organelles such as mitochondria and ribosomes in regions of thickened endothelium is consistent with activated endothelial cells. Luminal filopodia in male β3-null coronary capillaries appear to extend toward (F) or interact with (G-H) adjacent filopodia. The presence of electron-dense staining at points of contact suggests that interactions between filopodia may represent adhesive events. Connections between filopodia appear to form pockets within a single vessel lumen, consistent with angiogenesis by division of existing vessels. Bar = 500 nm.

Figure 5

Expanded vacuoles and intussusception in male β3-null coronary capillaries. Transmission electron micrographs show that coronary capillaries from male β3-null mice contain single endothelial cells with expanded vacuoles (A-D) that, like pockets formed by interacting filopodia, are consistent with angiogenesis by division of existing vessels. Some vessels have a limited luminal area and appear to be composed of a single endothelial cell (B), because cell-cell junctions are not visible. Although less frequent, some endothelial cells have luminal processes that extend across the lumen, resembling angiogenesis by intussusception (E). Asterisks indicate expanded vacuoles; arrows, intussusception. Bar = 500 nm.

Figure 6

Blood vessel phenotype in male β3-null mice is confirmed by scanning electron microscopy. Coronary capillaries from adult wild-type mice (A-B) have a smooth luminal surface consistent with images acquired using TEM. Freeze-fractured samples prepared for SEM reveal filopodia extending into the vessel lumen (C-D) or across the lumen to make adhesive contact with the opposite wall (E). Luminal divisions are visible, which may represent the expanded vacuoles observed via TEM (F). Arrows indicate filopodia; asterisk, expanded vacuole. Bar = 500 nm.

Figure 7

VEGF/Flk-1 activity contributes to immature vascular phenotype in male β3-null mice. Two hours following intravenous injection of VEGF into adult male wild-type mice, 22% of coronary capillaries examined exhibited luminal filopodia, extended vacuoles, and/or intussusception. These characteristics following VEGF injection are of a similar frequency as observed for untreated adult male β3-null mice. When adult male β3-null mice are treated for 2 days with pharmacologic inhibitors of either Flk-1 or VEGF (but not downstream signaling molecules such as Src kinases), the frequency of immature coronary capillaries is reduced to normal levels. These findings suggest that enhanced VEGF signaling through Flk is responsible for the vascular phenotype in adult male β3-null mice. Interestingly, VEGF injection into adult female wild-type or β3-null mice did not produce the angiogenic phenotype. Data represent mean ± SE.