Abstract 709: Aging Determines Different Osteochondrogenic Protein Expression Profile Associated With Increased Calcification and Elastocalcinosis in Diabetic Patients Submitted to Amputation

Abstract

Peripheral artery disease (PAD) frequently occurs in diabetes mellitus (DM) and associates with limb ischemia and amputation. Vascular calcification, which is very prevalent both in DM and in the elderly, contributes to PAD pathophysiology and further increases amputation risk. Our objectives were to quantify vascular calcification, elastic and collagen fiber content, besides osteochondrogenic protein expression in arteries from 2 diabetic patients at the extremes of age. Femoral and dorsalis pedis artery from a 92-year-old male (O) and a 40-year-old male (Y), both with DM, were harvested at the time of amputation caused by limb ischemia. Other cardiovascular risk factors were similar among patients. Specimens were fixed in buffered formaldehyde, processed and stained for hematoxylin-eosin, Verhoeff-van-Gieson and Masson’s trichrome. Immunohistochemical analysis was performed to assess osteogenic protein expression such as RUNX2, alkaline phosphatase (ALP) and Osterix (OSX), which are upregulated during vascular smooth muscle cell reprogramming to an osteogenic phenotype, responsible for hydroxyapatite deposition. Arteries from 92-year-old patient showed markedly architectural changes of the vascular wall in comparison with the younger patient sample, such as decreased collagen (O=0.8±0.2% versus Y=33.5±5.2% area/field, p<.05) and elastic fibers (O=1.6±0.4% vs. Y=7.8±1.5% area/field, p<.05) content. Coincidently, we demonstrated increased vascular calcification (O=209.7±27.5mm 2 /field vs. Y=30.6±6.3 mm 2 /field). Osteochondrogenic protein expression also showed opposing results: OSX stained around large calcification nuclei, mostly in the older, but not in the younger patient samples. Furthermore, RUNX2 and ALP expression, which determines early osteoblastic cell differentiation, occurred around small calcium deposits, mainly in the 40-year-old patient. We demonstrated that age and time-length exposure to diabetes are predictive of collagen and elastic fibers content associated with increased calcification area. Moreover, osteochondrogenic protein expression profile favors RUNX2 and ALP in the younger vs OSX in the older patient respectively, implying different stages of the disease.