Ren-Ming Hu

Vasoactive Peptides Modulate Vascular Endothelial Cell Growth Factor Production and Endothelial Cell Proliferation and Invasion

The proliferation of vascular endothelial cells (EC) is an important event in angiogenesis. The synthesis of the EC growth factor, vascular endothelial cell growth factor (VEGF), is stimulated by a variety of activators; but the effects of important vasoactive peptides are not well understood, and there are no known natural inhibitors of VEGF production. We found that the vasoactive peptides endothelin (ET)-1 and ET-3 stimulated the synthesis of VEGF protein 3–4-fold in cultured human vascular smooth muscle cells, comparable in magnitude to hypoxia. ET-1 and ET-3 acted through the ETA and ETB receptors, respectively, and signaling through protein kinase C was important. Atrial natriuretic peptide (ANP), C-type natriuretic peptide, and C-ANP-(4–23), a ligand for the natriuretic peptide clearance receptor, equipotently inhibited production of VEGF by as much as 88% and inhibited ET- or hypoxia-stimulated VEGF transcription. EC proliferation and invasion of matrix were stimulated by VEGF secreted into the medium by ET-incubated vascular smooth muscle cells. This was inhibited by ANP. Our results identify the natriuretic peptides as the first peptide inhibitors of VEGF synthesis and indicate a novel mechanism by which vasoactive peptides could modulate angiogenesis.

Insulin Stimulates Production and Secretion of Endothelin From Bovine Endothelial Cells

Endothelin, a vasoconstrictor peptide secreted from endothelial cells, has been thought to play a role in various forms of vascular disease. Diabetes mellitus is well known for its association with accelerated atherosclerosis and microvascular damage. Although the basis for the vessel insult is multifactorial, hyperinsulinemia is thought to contribute by an unknown mechanism. In this study, we sought to determine whether insulin stimulates the production and secretion of ET-1 as a possible basis for the association of hyperinsulinemia and vascular disease. We demonstrated that insulin significantly stimulates the gene expression and secretion of ET-1 from cultured BAEC, and that insulin increases ET-1 mRNA expressed in BBCEC. Insulin caused a maximal twofold inducement above control ET-1 mRNA expression in a dose-related fashion in BAEC. The increased mRNA resulted from increased transcription, as determined by nuclear run-off studies. Increased ET-1 mRNA was seen after 4 h of incubation with insulin: the peak occurred at 6–8 h and persisted for 24 h. Insulin caused as much as a fourfold stimulation of ET-1 secretion from BAEC in a dose-related fashion, including a twofold increase at a physiological concentration (10−9 M): The increase began at 1 h of incubation and continued for the entire 24-h incubation period. The insulin-induced increases in both ET-1 mRNA and ET-1 protein secretion were significantly attenuated by genistein, a tyrosine kinase inhibitor. This stimulation probably occurred through the insulin receptor, because IGF-1 had no effect on ET-1 gene expression or secretion from these cells. Actinomycin-D inhibited the stimulation of ET-1 mRNA by insulin, whereas cycloheximide caused a superinducement of insulins effect. Rats implanted with subcutaneous insulin pellets for 10 days had markedly elevated plasma ET levels, confirming a stimulatory role for insulin in vivo, in both diabetic and normal rodents. This study suggests that circulating hyperinsulinemia might induce the production and secretion of ET-1, a powerful endogenous vasoconstrictor and mitogen for the vascular smooth muscle cell. This interaction could underlie the increased vascular disease characteristic of hyperinsulinemic diabetic states.

Atrial and brain natriuretic peptides stimulate the production and secretion of C-type natriuretic peptide from bovine aortic endothelial cells.

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family which is produced in vascular endothelial cells and may play an important paracrine role in the vasaculature. We sought to determine the regulation of CNP production by other vasoactive peptides from cultured aortic endothelial cells. The vasoconstrictors endothelin-1 and angiotensin II had little effect on the basal secretion of CNP. In contrast, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) strongly stimulated the secretion of CNP. BNP caused as much as a 400-fold enhancement above the basal accumulated secretion of CNP over 24 h at a concentration of 1 microM; this was 20 times greater than the stimulatory effect of ANP, BNP and ANP also significantly enhanced the production of new CNP protein (translation) and mRNA expressed in the BAEC. In contrast, C-ANP-4-23, a truncated form of ANP which selectively binds to the natriuretic peptide clearance receptor, did not stimulate CNP secretion. The enhanced production and secretion of CNP, caused by either ANP or BNP, was significantly prevented by LY 83583, an inhibitor of cGMP generation, and was also attenuated by KT 5823, an inhibitor of cGMP-dependent protein kinase. Our results indicate that ANP and BNP can stimulate CNP production through a guanylate cyclase receptor on endothelial cells. BNP is a much more potent stimulator of CNP secretion, compared to ANP. Our findings suggest that the vasodilatory, and anti-mitogenic effects of ANP and BNP in the vasculature could occur in part through CNP production and subsequent action if these interactions occur in vivo.

High density lipoproteins stimulate the production and secretion of endothelin-1 from cultured bovine aortic endothelial cells.

The concentration of HDL in the blood inversely correlates with the incidence of cardiovascular disease, probably related to the ability of these lipoproteins to efflux cholesterol from vascular cells. it is also possible that HDL affect the production or action of vasoactive peptides implicated in the development of vascular diseases. Therefore, we determined the effects of human HDL on the production and secretion of endothelin-1 (ET-1) from cultured bovine aortic endothelial cells. HDL produced a highly significant stimulation of endothelin secretion (maximum 240% of control), even at very low levels of lipoproteins (1 microgram/ml). HDL also stimulated the translation of ET-1 by twofold in the bovine aortic endothelial cells. In contrast, HDL had no significant effect on steady state mRNA levels, transcript degradation, or transcription. Stimulation of ET-1 secretion by HDL was dependent on protein kinase C activation. Purified apo A-I, the major apoprotein of HDL, increased ET-1 secretion and translation approximately 85% as potently as HDL. Our results indicate that low concentrations of human HDL strongly stimulate the production of ET-1, a powerful vasoconstrictor and mitogen for the vascular smooth muscle cell. We propose that HDL may participate in the regulation of vasomotor tone through this potentially important effect in the vasculature.

Astrocyte Progression from G1 to S Phase of the Cell Cycle Depends upon Multiple Protein Interaction

The proliferation of cultured astrocytes is positively and negatively regulated, respectively, by the endogenous neuropeptides, endothelin-3 (ET-3) and atrial natriuretic peptide (ANP). Here, we determined the important steps for the modulation by ET and ANP of G1 to S phase cell cycle progression. ET-3 stimulated an increased number of fetal rat diencephalic astrocytes to progress through G1/S, and this was blocked significantly by ANP. ET augmented the gene expression and/or protein production of D-type, A and E cyclins, whereas ANP inhibited these events significantly. ET also stimulated the activation of the cyclin-dependent kinases Cdk2, Cdk4, and Cdk6, directed against the retinoblastoma protein pRb, and this was inhibited by as much as 80% by ANP. As an additional mechanism of cell cycle restraint, ANP stimulated the production of multiple cyclin-dependent kinase inhibitory (CKI) proteins, including p16, p27, and p57. This was critical because antisense oligonucleotides to each CKI reversed ANP-induced inhibition of ET-stimulated DNA synthesis by as much as 85%. CKI antisense oligonucleotides also reversed the ANP inhibition of Cdk phosphorylation of pRb. In turn, ET inhibited ANP-stimulated production of the CKIs, thereby promoting cell cycle progression. Specific and changing associations of the CKI with Cdk2 and Cdk4 were stimulated by ANP and inhibited by ET. Our findings identify several mechanisms by which endogenous modulators of astrocyte proliferation can control the G1-S progression and indicate that multiple CKIs are necessary to restrain cell cycle progression in these cells.