Microvesicles, skin microcirculation and clinical microangiopathy in type 1 diabetes

• Background: Type 1 diabetes is a proinflammatory and prothrombotic disease associated with a highly elevated risk of microvascular complications, including nephropathy, retinopathy, and neuropathy, as well as premature cardiovascular disease. Aim: this thesis sought to study plasma microvesicles (MVs) and skin microcirculation in relation to clinical microangiopathy in type 1 diabetes, to try to identify potential clinical biomarkers. In a separate laboratory investigation, we explored how different centrifugation protocols affect flow cytometry measurement of MVs. Methods: In Papers I and II we measured plasma MVs of different cellular origins and their expression of procoagulant phosphatidylserine (PS) and proinflammatory HMGB1 in 236 well-characterized patients and 100 matched healthy controls using flow cytometry. Plasma lactadherin was analyzed using ELISA. In Paper III we compared the MISEV 2018 centrifugation protocol to different single centrifugation protocols in terms of effect on MV levels and remaining cell fragments. In Paper IV we investigated skin microvascular reactivity in 61 patients with type 1 diabetes with and without microangiopathy and 31 healthy controls. Results: In Paper I we found significantly higher levels of total plasma MVs, PS+ and PSMVs in patients compared to controls, but with no correlation to clinical microangiopathy. Plasma lactadherin was increased in patients versus controls, and patients with microangiopathy had significantly higher levels than patients without. In Paper II, we found that subpopulations of both endothelial and platelet derived MVs were significantly increased in patients compared to controls. We also showed a significant increase in HMGB1+ MVs in type 1 diabetes. In Paper III, we demonstrated that a single round of centrifugation prior to freezing, followed by re-centrifugation after thawing, resulted in lower MV levels but also lower cell fragmentation than the MISEV 2018 protocol, decreasing the likelihood of false positive events. In Paper IV, we found that impairment in skin microvascular reactivity in patients with type 1 diabetes related to their degree of clinical microangiopathy. Conclusions: Type 1 diabetes is associated with significantly increased MV levels, but with no relationship to clinical microangiopathy. The striking elevation of endothelial MV levels in type 1 diabetes points to pervasive endothelial dysfunction, whereas evidence of platelet hyperactivity was less pronounced. Elevated HMGB1+ MVs and plasma lactadherin in patients with type 1 diabetes could have implications for future vascular complications. A standardized centrifugation protocol using a single round of centrifugation prior to freezing, followed by recentrifugation prior to flow cytometry analysis, was found to be a pragmatic approach for measuring MVs in larger clinical studies. Skin microvascular reactivity correlated with the severity of clinical microangiopathy in patients, suggesting that type 1 diabetes seems to affect the microvasculature in the whole body. Our findings support the use of skin microcirculation as a clinically relevant bedside model for the study of microangiopathy.

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