Amylin-amide displays a proliferative effect on human umbilical vein endothelial cells

Amylin, also known as diabetes-associated peptide and islet amyloid polypeptide, is a 37 amino acid peptide which was recently isolated from pancreatic amyloids of type I1 diabetics [ I ] . Amylin shares 46% amino acid sequence identity with calcitonin gene-related peptide (CGRP) [2]. The human amylin gene is located on the short arm of chromosome 12 131 and may share a common origin with the genes for CGRP, insulin and insulin-like growth factors. The latter peptides have been assigned to human chromosome 11 [4]; both chromosomes 1 1 and 12 are thought to be derived from a common ancestral gene [S]. In view of the findings that CGRP and amylin share a common binding site on human osteosarcoma cells [6], and the fact that CGRP possesses a proliferative effect [ 71, we have examined the growth-factor-like effects of amylin-amide on human umbilical vein endothelial cells. Umbilical vein endothelial cells were isolated as described elsewhere [ 81. Fresh human umbilical veins were thoroughly rinsed with phosphate-buffered saline (37"C), filled with solution containing 0.1% (w/v) collagenase and incubated for 10 min. Cells were harvested by gentle centrifugation at 500 g for 3 min and resuspended in medium 199 (M 199; Gibco) containing fetal calf serum (20%, v/v, heat inactivated), and antibiotics (penicillin 50 pg/ml; streptomycin 50 units/rnl). The cells were grown on fibronectin-coated plates (2 pg/ cm') and their viability was assessed by measuring the production of factor VI11-related antigen and cell proliferation. Cells were plated at 4 x 1U3/cm? and, after overnight growth, the cells were growth-arrested by being grown in serum-free medium. The growth-arrested cells were subsequently exposed to amylin-amide, CGRP, eel-calcitonin, porcine-insulin, met-enkephalin, substance P, and somatostatin at a range o f concentrations (0 .1 nM100 nM) in M 199 containing So/" (v /v) FCS. The rate of increase in the number of endothelial cells was determined after six days of exposure to the peptides, the medium containing the appropriate peptides being renewed after 24 h. The cells used for DNA synthesis were also serum-starved overnight and the incorporation of S-bromo-2'-deoxyuridine was measured by the e.1.i.s.a. method with monoclonal antibodies following 2 h of exposure to various peptides in M I 9 9 containing FCS (S%, v/v). The cells grown in M I99 containing FCS (20"/0, v/v) were used as controls. Insulin was found to increase cell number by 7 1% f 8% ( P < 0.00 1 ), but met-enkephalin, substance P, and somatostatin were found t o be without proliferative effect. Amylinamide ( 1 0 nM), CGRP ( 1 n M ) and eel-calcitonin ( 1 nM) caused a significant increase in the cell numbers; 56% k 14"/0 ( P < O . O I ) , SO'%)f9%) (Pdeoxyuridine, was significantly