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Background and aims Arteriogenesis, the positive outward remodeling and growth of pre-existent collateral vessels, holds potential as a novel treatment for ischemic vascular disease. An extracranial arteriogenesis model in a pig will allow us to study molecular changes in a complex arteriolar network in a more clinically relevant large-animal model. To increase fluid shear stress in the brain, an experimental carotid arteriovenous fistula (AVF model) in minipigs was established, providing high flow through the extracranial rete mirabile. The aim of the study was to examine whether creation of a carotid AVF can induce extracranial arteriogenesis in the pig. Methods Angiography was performed to demonstrate blood flow diversion. Animals were sacrificed after 0, 3 and 14 days post-surgery and both retia mirabilia were removed. Immunohistochemical analysis was performed to analyze cell proliferation and accumulation of mononuclear cells in the vessel wall. Results After 3 days of high-flow conditions, increases in vascular cell proliferation (approximately 1.5-fold; p = 0.143) and monocyte invasion (approximately 6-fold; p = 0.057) were observed when compared to animals sacrificed immediately after AVF formation. Quantitative PCR (RT-qPCR) analysis from rete mirabile tissue samples 3 days post-surgery revealed that monocyte chemoattractant protein (MCP)-1 and tissue inhibitor of metalloproteinases (TIMP)-1 were highly upregulated. Expression of the pro-arteriogenic marker, CD44, reached maximum expression level 14 days post-surgery. Conclusions In response to high levels of shear stress produced in the pig AVF model, the onset of the arteriogenic process can be induced. This was demonstrated by enhanced cell proliferation, monocyte invasion and vascular remodeling.

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.

Even tissues capable of complete regeneration, such as bone, show an age-related reduction in their healing capacity. Here, we hypothesized that this decline is primarily due to cell non-autonomous (extrinsic) aging mediated by the systemic environment. We demonstrate that culture of mesenchymal stromal cells (MSCs) in serum from aged Sprague–Dawley rats negatively affects their survival and differentiation ability. Proteome analysis and further cellular investigations strongly suggest that serum from aged animals not only changes expression of proteins related to mitochondria, unfolded protein binding or involved in stress responses, it also significantly enhances intracellular reactive oxygen species production and leads to the accumulation of oxidatively damaged proteins. Conversely, reduction of oxidative stress levels in vitro markedly improved MSC function. These results were validated in an in vivo model of compromised bone healing, which demonstrated significant increase regeneration in aged animals following oral antioxidant administration. These observations indicate the high impact of extrinsic aging on cellular functions and the process of endogenous (bone) regeneration. Thus, addressing the cell environment by, for example, systemic antioxidant treatment is a promising approach to enhance tissue regeneration and to regain cellular function especially in elderly patients.