The Fibrin Matrix Regulates Angiogenic Responses within the Hemostatic Microenvironment through Biochemical Control

Abstract

Conceptually, premature initiation of post-wound angiogenesis could interfere with hemostasis, as it relies on fibrinolysis. The mechanisms facilitating orchestration of these events remain poorly understood, however, likely due to limitations in discerning the individual contribution of cells and extracellular matrix. Here, we designed an in vitro Hemostatic-Components-Model (HCM) to investigate the role of the fibrin matrix as protein factor-carrier, independent of its cell-scaffold function. After characterizing the proteomic profile of HCM-harvested matrix releasates, we demonstrate that the key pro-/anti-angiogenic factors, VEGF and PF4, are differentially bound by the matrix. Changing matrix fibrin mass consequently alters the balance of releasate factor concentrations, with differential effects on basic endothelial cell (EC) behaviors. While increasing mass, and releasate VEGF levels, promoted EC chemotactic migration, it progressively inhibited tube formation, a response that was dependent on PF4. These results indicate that the clot’s matrix component initially serves as biochemical anti-angiogenic barrier, suggesting that post-hemostatic angiogenesis follows fibrinolysis-mediated angiogenic disinhibition. Beyond their significance towards understanding the spatiotemporal regulation of wound healing, our findings could inform the study of other pathophysiological processes in which coagulation and angiogenesis are prominent features, such as cardiovascular and malignant disease.

Comparative Evaluation of the Angiogenic Potential of Hypoxia Preconditioned Blood-Derived Secretomes and Platelet-Rich Plasma: An In Vitro Analysis

“…Subsequently, the filter was removed and the capped syringe was placed upright in an incubator (37 • C/5% CO 2 ) and incubated for 4 or 7 days (blood incubation time), without any prior centrifugation. Pericellular local hypoxia (~1% O 2 ) was induced in situ through cell-mediated O 2 consumption, by controlling the blood volume per unit area (BVUA > 1 mL/cm 2 ), and consequently the PBC seeding density in the blood container [13,19]. After the predefined incubation time, the blood was passively separated into three layers, from top to bottom; plasma/serum, clot/buffy coat, red blood cell component, so that the top layer comprising hypoxia preconditioned plasma or serum (HPP/HPS) could be filtered (0.2 µm pore filter, Sterifix ® , B Braun AG, Germany) into a new syringe (see Figure 1B), removing cells/cellular debris.…”

“…Hypoxia preconditioned blood-derived secretomes can be generated through the process of "extracorporeal wound simulation", which entails peripheral blood incubation under physiological temperature (37 • C) and hypoxia (1-10% O 2 ) ( Figure 1B) [2,5,19,44]. As previously demonstrated, pericellular hypoxia can be achieved in situ within the blood incubation chamber, by adjusting the blood volume per unit area (BVUA), i.e., the PBC seeding density, and hence cellular O 2 consumption, thus overcoming the need for an oxygen-controlling incubator [2,13,19]. It is already established that PBCs respond to stress (e.g., hypoxia, ischemia, inflammation, ultrasound) by upregulating a range of pro-angiogenic growth factors such as VEGF [2,5,[45][46][47][48], bFGF [46][47][48], IL-8 [2,47,48] and MMP-9 [2,47].…”

“…During the haemostatic phase, platelets play a paramount role by concentrating at the site of injury, and releasing a plethora of growth factors through degranulation [8][9][10][11]. The clot fibrin matrix thereafter provides a scaffold for migrating peripheral blood cells (PBCs), e.g., neutrophils, macrophages, lymphocytes, and other cell types (e.g., fibroblasts, endothelial cells) [12][13][14]. As a result of vascular trauma and the consequent disruption in oxygen supply, PBCs are exposed to local hypoxia, which leads to the production and release of pro-angiogenic growth factors that stimulate new vessel formation and reestablishment of the wound bed's microcirculation [15][16][17][18].…”

“…Biomedicines 2019, 7, x FOR PEER REVIEW 3 of 23 [2,13,19]. It is already established that PBCs respond to stress (e.g., hypoxia, ischemia, inflammation, ultrasound) by upregulating a range of pro-angiogenic growth factors such as VEGF [2,5,[45][46][47][48], bFGF [46][47][48], IL-8 [2,47,48] and MMP-9 [2,47].…”

The Use of Blood Components in Tissue Engineering

“…Известно, что фибриноген может связывать многие биологически активные белки, такие как фактор роста фибробластов (FGF), фактор роста тромбоцитов (PDGF), трансформирующий ростовой фактор бета (TGF-β), тромбоспондин-1 (TSP1), интерликин-1(IL1), которые высвобождаются после его полимеризации [24-27]. В работе E. Hadjipanayi et al [28] показано, что фактор роста эндотелия сосудов (VEGF) и тромбоцитарный фактор (PF4), являющиеся ключевыми про-и антиангиогенными факторами, дифференциально связаны с матрицей фибрина. При этом матрица может осуществлять биохимический контроль ангиогенеза путем уравновешивания относительных концентраций и контролируемого массзависимого высвобождения про-и антиангиогенных факторов.…”