Adipose tissue-derived microvascular fragments are promising vascularisation units for applications in the field of tissue engineering. Elderly patients are the major future target population of such applications due to an increasing human life expectancy. Therefore, we herein investigated the effect of aging on the fragments' vascularisation capacity. Microvascular fragments were isolated from epididymal fat pads of adult (8 months) and aged (16 months) C57BL/6 donor mice. These fragments were seeded onto porous polyurethane scaffolds, which were implanted into dorsal skinfold chambers to study their vascularisation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with fragments from aged donors exhibited a significantly lower functional microvessel density and intravascular blood flow velocity. This was associated with an impaired vessel maturation, as indicated by vessel wall irregularities, constantly elevated diameters and a lower fraction of CD31/α-smooth muscle actin double positive microvessels in the implants' border and centre zones. Additional in vitro analyses revealed that microvascular fragments from adult and aged donors do not differ in their stem cell content as well as in their release of angiogenic growth factors, survival and proliferative activity under hypoxic conditions. However, fragments from aged donors exhibit a significantly lower number of matrix metalloproteinase-9-positive perivascular cells. Taken together, these findings demonstrate that aging is a crucial determinant for the vascularisation capacity of isolated microvascular fragments.

The seeding of scaffolds with adipose tissue-derived microvascular fragments represents a promising strategy to establish a sufficient blood supply in tissue constructs. Herein, we analysed whether a single application of macrophage-activating lipopeptide-2 (MALP-2) at the implantation site further improves the early vascularisation of such microvessel-seeded constructs. Microvascular fragments were isolated from epididymal fat pads of C57BL/6 mice. The fragments were seeded on polyurethane scaffolds, which were implanted into mouse dorsal skinfold chambers exposed to MALP-2 or vehicle (control). The inflammatory host tissue response and the vascularisation of the scaffolds were analysed using intravital fluorescence microscopy, histology and immunohistochemistry. We found that the numbers of microvascular adherent leukocytes were significantly increased in MALP-2-treated chambers during the first 3 days after scaffold implantation when compared to controls. This temporary inflammation resulted in an improved vascularisation of the host tissue surrounding the implants, as indicated by a higher density of CD31-positive microvessels at day 14. However, the MALP-2-exposed scaffolds themselves presented with a lower functional microvessel density in their centre. In addition, in vitro analyses revealed that MALP-2 promotes apoptotic cell death of endothelial and perivascular cells in isolated microvascular fragments. Hence, despite the beneficial pro-angiogenic properties of MALP-2 at the implantation site, the herein evaluated approach may not be recommended to improve the vascularisation capacity of microvascular fragments in tissue engineering applications.

BackgroundInflammation strongly contributes to atherosclerosis initiation and progression. Consequently, recent clinical trials pharmacologically targeted vascular inflammation to decrease the incidence of atherosclerosis-related complications. Colchicine, a microtubule inhibitor with anti-inflammatory properties, reduced cardiovascular events in patients with recent acute coronary syndrome and chronic coronary disease. However, the biological basis of these observations remains elusive. We sought to explore the mechanism by which colchicine beneficially alters the course of atherosclerosis.Methods and ResultsIn mice with early atherosclerosis (Apoe-/- mice on a high cholesterol diet for 8 weeks), we found that colchicine treatment (0.25 mg/kg bodyweight once daily over four weeks) reduced numbers of neutrophils, inflammatory monocytes and macrophages inside atherosclerotic aortas using flow cytometry and immunohistochemistry. Consequently, colchicine treatment resulted in a less inflammatory plaque composition and reduced plaque size. We next investigated how colchicine prevented plaque leukocyte expansion and found that colchicine treatment mitigated recruitment of blood neutrophils and inflammatory monocytes to plaques as revealed by adoptive transfer experiments. Causally, we found that colchicine reduced levels of both leukocyte adhesion molecules and receptors for leukocyte chemoattractants on blood neutrophils and monocytes. Further experiments showed that colchicine treatment reduced vascular inflammation also in post-myocardial infarction accelerated atherosclerosis through similar mechanisms as documented in early atherosclerosis. When we examined whether colchicine also decreased numbers of macrophages inside atherosclerotic plaques by impacting monocyte/macrophage transitioning or in-situ proliferation of macrophages, we report that colchicine treatment did not influence macrophage precursor differentiation or macrophage proliferation using cell culture experiments with bone marrow derived macrophages.ConclusionsOur data reveal that colchicine prevents expansion of plaque inflammatory leukocytes through lowering recruitment of blood myeloid cells to plaques. These data provide novel mechanistic clues on the beneficial effects of colchicine in the treatment of atherosclerosis and may inform future anti-inflammatory interventions in patients at risk.

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