The role of Multimerin-2 in pericyte recruitment and vascular stability

The proper function of normal blood vessels is ensured by endothelial cells (ECs), which are tightly connected to one another by specific junctions, thus regulating vessel permeability and the intra- and extra-vasation of immune cells. Additional stability regulators are pericytes and the vessels’ basement membrane (BM). Specifically, pericytes are mural cells embedded in the BM of blood microvessels, and envelope the vessels promoting vessel stabilization and maturation. In the context of vascular homeostasis, we demonstrated that Multimerin-2, an extracellular matrix glycoprotein secreted by ECs and deposited along all blood vessels, is key in maintaining the integrity of ECs junctions. Given its strategic localization in juxtaposition between the endothelium and pericytes and the finding that retinal vessels from Multimerin-2-/- animals display impaired pericytes coverage, the aim of the present project was to investigate whether Multimerin-2 affected pericytes biology and if its loss impacted on the vascular efficiency and immune cell infiltration. We found that Multimerin-2 affected pericyte recruitment to the newly formed vessels both directly and indirectly. Indeed, we demonstrated that pericytes efficiently migrate towards a Multimerin-2 gradient and that ECs challenged with Multimerin-2 displayed an increased expression of PDGF-BB and HB-EGF, two cytokines that foster pericyte recruitment. Moreover, we showed that Multimerin-2 functions as an adhesion substrate for pericytes, indicating that the expression of this molecule promotes both the recruitment and the pericyte-endothelial cell interaction. Once pericytes reach the endothelium, they need to proliferate to properly embrace the vasculature and we unveiled that Multimerin-2 increases pericyte viability and proliferation, hinging on IGF1R-activation. Our in vitro results were further corroborated in vivo exploiting the Multimerin-2-/- mouse model. Tumors developed in Multimerin-2-/- mice following the injection of the syngeneic high grade serous ovarian cancer cells ID8 Trp53-/- Pten-/- and the colon carcinoma cells MC-38 showed impaired pericyte recruitment to the vessels and increased intratumoral hypoxia. Furthermore, in accordance with the notion that vascular inefficiency negatively affects drug delivery, following intraperitoneal injection of the drug, tumors grown in Multimerin-2-/- mice displayed a reduced number of cisplatin-positive nuclei. Moreover, to verify if loss of Multimerin-2 impacted on the immune response we carried out a preliminary characterization of the tumor-infiltrating immune cells. Interestingly, we found that tumors from Multimerin-2-/- mice display increased recruitment of macrophages. This finding was further supported by in vitro analyses showing that a higher number of THP-1 cells, monocytic-like cell line, transmigrated across the Multimerin-2-depleted endothelium. Taken together, these results suggest that Multimerin-2 expression is required for proper recruitment, proliferation, and adhesion of pericytes to the endothelium. As a consequence, upon loss of Multimerin-2 the endothelium is destabilized leading to increased intratumoral hypoxia and poor drug delivery. Importantly, loss of Multimerin-2 also affects the immune response suggesting that the outcome of the patients may significantly be affected depending on the levels of Multimerin-2 loss in the tumor patients.

The proper function of normal blood vessels is ensured by endothelial cells (ECs), which are tightly connected to one another by specific junctions, thus regulating vessel permeability and the intra- and extra-vasation of immune cells. Additional stability regulators are pericytes and the vessels’ basement membrane (BM). Specifically, pericytes are mural cells embedded in the BM of blood microvessels, and envelope the vessels promoting vessel stabilization and maturation. In the context of vascular homeostasis, we demonstrated that Multimerin-2, an extracellular matrix glycoprotein secreted by ECs and deposited along all blood vessels, is key in maintaining the integrity of ECs junctions. Given its strategic localization in juxtaposition between the endothelium and pericytes and the finding that retinal vessels from Multimerin-2-/- animals display impaired pericytes coverage, the aim of the present project was to investigate whether Multimerin-2 affected pericytes biology and if its loss impacted on the vascular efficiency and immune cell infiltration. We found that Multimerin-2 affected pericyte recruitment to the newly formed vessels both directly and indirectly. Indeed, we demonstrated that pericytes efficiently migrate towards a Multimerin-2 gradient and that ECs challenged with Multimerin-2 displayed an increased expression of PDGF-BB and HB-EGF, two cytokines that foster pericyte recruitment. Moreover, we showed that Multimerin-2 functions as an adhesion substrate for pericytes, indicating that the expression of this molecule promotes both the recruitment and the pericyte-endothelial cell interaction. Once pericytes reach the endothelium, they need to proliferate to properly embrace the vasculature and we unveiled that Multimerin-2 increases pericyte viability and proliferation, hinging on IGF1R-activation. Our in vitro results were further corroborated in vivo exploiting the Multimerin-2-/- mouse model. Tumors developed in Multimerin-2-/- mice following the injection of the syngeneic high grade serous ovarian cancer cells ID8 Trp53-/- Pten-/- and the colon carcinoma cells MC-38 showed impaired pericyte recruitment to the vessels and increased intratumoral hypoxia. Furthermore, in accordance with the notion that vascular inefficiency negatively affects drug delivery, following intraperitoneal injection of the drug, tumors grown in Multimerin-2-/- mice displayed a reduced number of cisplatin-positive nuclei. Moreover, to verify if loss of Multimerin-2 impacted on the immune response we carried out a preliminary characterization of the tumor-infiltrating immune cells. Interestingly, we found that tumors from Multimerin-2-/- mice display increased recruitment of macrophages. This finding was further supported by in vitro analyses showing that a higher number of THP-1 cells, monocytic-like cell line, transmigrated across the Multimerin-2-depleted endothelium. Taken together, these results suggest that Multimerin-2 expression is required for proper recruitment, proliferation, and adhesion of pericytes to the endothelium. As a consequence, upon loss of Multimerin-2 the endothelium is destabilized leading to increased intratumoral hypoxia and poor drug delivery. Importantly, loss of Multimerin-2 also affects the immune response suggesting that the outcome of the patients may significantly be affected depending on the levels of Multimerin-2 loss in the tumor patients.