Vera Jankowski

Metabolic stimuli, pressure, and fluid shear stress (FSS) are major mediators of vascular plasticity. The exposure of the vessel wall to increased laminar FSS is the main trigger of arteriogenesis, the remodelling of pre-existent arterio-arteriolar anastomoses to functional conductance arteries. In this study, we have used an in vitro bioreactor to investigate cell-specific interactions, molecular mechanisms as well as time-dependent effects under laminar FSS conditions. This bioreactor termed “artificial artery” can be used for screening potential arterio-protective substances, pro-arteriogenic factors, and for investigating biomarkers of cardiovascular diseases such as cardiac diseases. The bioreactor is built up out of 14 hollow fiber membranes colonized with endothelial cells (HUVECs) on the inside and smooth muscle cells (HUASMCs) on the outside. By means of Hoechst 33342 staining as well as immunocytochemistry of ß-catenin and α-smooth-muscle-actin, a microporous polypropylene membrane was characterized as being the appropriate polymer for co-colonization. Defined arterial flow conditions (0.1 N/m2 and 3 N/m2), metabolic exchange, and cross-talk of HUVECs and HUASMCs through hollow fibers mimic physiological in vivo conditions of the vasculature. Analysing mono- and co-culture secretomes by MALDI-TOF-TOF mass spectrometry, we could show that HUVECs secreted Up4A upon 3 N/m2. A constant cellular secretion of randomly chosen peptides verified viability of the “artificial artery” for a cultivation period up to five days. qRT-PCR analyses revealed an up-regulation of KLF2 and TIMP1 as mechano-regulated genes and demonstrated arterio-protective, homeostatic FSS conditions by a down-regulation of EDN1. Expression analyses of VWF and EDN1 furthermore confirmed that RNA of both cell types could separately be isolated without cross-contamination. CCND1 mRNA expression in HUVECs did not change upon FSS indicating a quiescent endothelial phenotype. Taken together, the “artificial artery” provides a solid in vitro model to test pharmacological active compounds for their impact on arterio-damaging or arterio-protective properties on vascular response.

Dinucleoside polyphosphates are well described as direct vasoconstrictors and as mediators with strong proliferative properties, however, less is known about their effects on nucleotide-converting pathways. Therefore, the present study investigates the effects of Ap₄A (diadenosine tetraphosphate), Up₄A (uridine adenosine tetraphosphate) and Ap₅A (diadenosine pentaphosphate) and the non-selective P2 antagonist suramin on human serum and endothelial nucleotide-converting enzymes. Human serum and HUVECs (human umbilical vein endothelial cells) were pretreated with various concentrations of dinucleotide polyphosphates and suramin. Adenylate kinase and NDP kinase activities were then quantified radiochemically by TLC analysis of the ATP-induced conversion of [³H]AMP and [³H]ADP into [³H]ADP/ATP and [³H]ATP respectively. Endothelial NTPDase (nucleoside triphosphate diphosphohydrolase) activity was additionally determined using [³H]ADP and [³H]ATP as preferred substrates. Dinucleoside polyphosphates and suramin have an inhibitory effect on the serum adenylate kinase [pIC₅₀ values (−log IC₅₀): Ap₄A, 4.67±0.03; Up₄A, 3.70±0.10; Ap₅A, 6.31±0.03; suramin, 3.74±0.07], as well as on endothelial adenylate kinase (pIC₅₀ values: Ap₄A, 4.17±0.07; Up₄A, 2.94±0.02; Ap₅A, 5.97±0.04; suramin, 4.23±0.07), but no significant effects on serum NDP kinase, emphasizing the selectivity of these inhibitors. Furthermore, Ap₄A, Up₄A, Ap₅A and suramin progressively inhibited the rates of [³H]ADP (pIC₅₀ values: Ap₄A, 3.38±0.09; Up₄A, 2.78±0.06; Ap₅A, 4.42±0.11; suramin, 4.10±0.07) and [³H]ATP (pIC₅₀ values: Ap₄A, 3.06±0.06; Ap₅A, 3.05±0.12; suramin, 4.14±0.05) hydrolyses by cultured HUVECs. Up₄A has no significant effect on the endothelial NTPDase activity. Although the half-lives for Ap₄A, Up₄A and Ap₅A in serum are comparable with the incubation times of the assays used in the present study, secondary effects of the dinucleotide metabolites are not prominent for these inhibitory effects, since the concentration of metabolites formed are relatively insignificant compared with the 800 μmol/l ATP added as a phosphate donor in the adenylate kinase and NDP kinase assays. This comparative competitive study suggests that Ap₄A and Ap₅A contribute to the purinergic responses via inhibition of adenylate-kinase-mediated conversion of endogenous ADP, whereas Up₄A most likely mediates its vasoregulatory effects via direct binding-mediated mechanisms.