The formation of blood vessels during vertebrate embryogenesis is required for normal development of the organism. Eelucidating the mechanisms regulating the proliferation and differentiation of EC and SMC the cell types which comprise both capillaries and larger vessels is crucial to understanding vascular morphogenesis. Cell number in tissues a governed by the balance between proliferation and programmed cell death (apoptosis). Many external signals have been shown to influence EC apoptosis, including inducers like tumor necrosis factor-alpha and inhibitor like vascular endothelial growth factor (VEGF). The intracellular signaling pathways involved in apoptosis have come under close scrutiny recently; in particular, activation of the Akt family of kinases has been strongly implicated protecting neuronal and fibroblast cells against apoptosis. However, no information is presently available on the role of Art kinases in EC. Based on the preliminary data which demonstrates the presence of Art kinases in EC and the upregulation of Akt kinase activity by VEGF, it is hypothesized that the apoptosis- protective effect of VEGF and other mitogens/survival factors on EC is due to activation of Akt.
Specific Aim 1 examines this hypothesis by a) using localizing antibodies to determine the spatial and temporal pattern of Akt expression in the developing mouse; b) measuring Akt kinase kinase activity in cultured EC exposed to apogenic stimuli, apoptosis- protectors, and combinations of the two; and c) forced expression, dominant-negative, and antisense oligonucleotide approaches to assess the functional role of Akt in cultured EC. Vascular morphogenesis (remodeling) also requires careful control of SMC proliferation. Based on work in the PI's laboratory and by others suggesting that heparan sulfates (including heparin) may be physiologic regulators of SMC proliferation in neonates and adults, it is hypothesized that these glycosaminoglycans are important in vascular morphogenesis. A corollary to this hypothesis is that SMC deficient in their response to heparin will display problems in arterial wall development. This appears to be the case in primary pulmonary hypertension of the newborn (one of the most common cardiovascular defects in human neonates), and in the Spontaneously Hypertensive Rat, a well-characterized model system for hypertension.
Specific Aim 2 examines this hypothesis by a) assessing the heparin-responsive phenotypes of embryonic and neonatal SMC; b) using in situ hybridization and localizing antibodies to heparin-induced CCN- like protein (HICP), a molecular marker for the heparin-responsive phenotype, to ascertain when and where in development the heparin- responsive phenotype appears; and c) examining the functional role of HICP in knock-out mice. The experiments proposed in these two aims should provide novel and important information about the cellular and molecular mechanisms that regulate formation of the vascular system in development.
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