Decreased S-nitrosylation of tissue transglutaminase contributes to age-related increases in vascular stiffness.

RATIONALE: Although an age-related decrease in NO bioavailability contributes to vascular stiffness, the underlying molecular mechanisms remain incompletely understood. We hypothesize that NO constrains the activity of the matrix crosslinking enzyme tissue transglutaminase (TG2) via S-nitrosylation in young vessels, a process that is reversed in aging. OBJECTIVE: We sought to determine whether endothelium-dependent NO regulates TG2 activity by S-nitrosylation and whether this contributes to age-related vascular stiffness. METHODS AND RESULTS: We first demonstrate that NO suppresses activity and increases S-nitrosylation of TG2 in cellular models. Next, we show that nitric oxide synthase (NOS) inhibition leads to increased surface and extracellular matrix-associated TG2. We then demonstrate that endothelium-derived bioactive NO primarily mediates its effects through TG2, using TG2(-/-) mice chronically treated with the NOS inhibitor l-N(G)-nitroarginine methyl ester (L-NAME). We confirm that TG2 activity is modulated by endothelium-derived bioactive NO in young rat aorta. In aging rat aorta, although TG2 expression remains unaltered, its activity increases and S-nitrosylation decreases. Furthermore, TG2 inhibition decreases vascular stiffness in aging rats. Finally, TG2 activity and matrix crosslinks are augmented with age in human aorta, whereas abundance remains unchanged. CONCLUSIONS: Decreased S-nitrosylation of TG2 and increased TG activity lead to enhanced matrix crosslinking and contribute to vascular stiffening in aging. TG2 appears to be the member of the transglutaminase family primarily contributing to this phenotype. Inhibition of TG2 could thus represent a therapeutic target for age-associated vascular stiffness and isolated systolic hypertension.

Glomerular protein levels of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 are lower in diabetic subjects.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix turnover throughout the body, including in renal glomeruli. We investigated protein levels of multiple MMPs (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 in glomeruli and investigated whether disease phenotypes were associated with levels of these proteins. Renal cortex was collected from 100 adult autopsy subjects arrayed across 17 tissue microarrays. Immunohistochemical staining intensity for each MMP and TIMP-1 was determined using quantitative color deconvolution techniques. We observed significantly decreased glomerular MMP-1 and TIMP-1 staining in subjects with diabetes, hypertension, and an estimated glomerular filtration rate <30 ml/min/1.73 m(2) in univariate analyses. MMP-1 staining, but not TIMP-1 staining, was inversely correlated with increased glomerular fibrosis (r = -0.40). In multivariable analysis, diabetes was robustly associated with decreased staining intensity. This study indicates that in human subjects, the long-term sequelae of diseases such as diabetes that cause chronic renal failure result in decreased TIMP-1 and MMP-1 proteins in renal glomeruli. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Use of human vascular tissue microarrays for measurement of advanced glycation endproducts.

Advanced glycation endproducts (AGEs) are present in the vasculature and are associated with vascular disease. We determined levels of AGEs in eight distinct adult vascular tissues using tissue microarray (TMA) technology and associated these levels with clinical characteristics. Medium-to-large caliber blood vessels were harvested from 100 adult autopsies to create 17 TMAs. AGE levels were evaluated by IHC using a polyclonal anti-AGE antibody on over 700 unique blood vessels. Slides were digitally scanned, and quantitative analysis was performed using a color deconvolution image analysis technique. Medial AGE staining was strongly correlated between all eight blood vessels. In the media, AGE staining levels were significantly higher at older ages (p=0.009), in white subjects (p<0.001) and with longer postmortem interval (PMI; p<0.0001). These associations remained significant after simultaneous adjustment for age, race/ethnicity, PMI, and diabetes status. Diabetes was associated with elevated AGE levels but only after adjustment for confounding by clinical variables including race/ethnicity, hypertension, and kidney function. This extensive vascular study shows that AGE accumulation in the macrovasculature is a global process affecting atherosclerosis-prone and -resistant vessels. It also suggests ethnicity has a previously undescribed role in vascular tissue AGE levels. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Tissue inhibitor of matrix metalloproteinase-3 levels in the extracellular matrix of lung, kidney, and eye increase with age.

Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) is an important regulator of matrix metalloproteinase activity in many types of disease, including atherosclerosis, neoplasia, and inflammatory conditions. Among TIMPs, TIMP-3 uniquely binds the extracellular matrix (ECM). We performed IHC staining on 17 tissue microarrays containing >1500 samples to determine the location of ECM TIMP-3 staining in a variety of predominantly vascular tissues. We found a unique pattern of TIMP-3 staining in the ECM of renal arterioles, small pulmonary vessels and parenchyma, and Bruch's membrane in the retina. There was no staining in larger caliber arteries including coronary and internal mammary arteries. TIMP-3 protein accumulation was found to be an age-dependent phenomenon, with staining appearing in all three tissues in early adulthood and becoming more robust among the elderly. These findings may help to explain the late onset of the TIMP-3-associated ocular diseases Sorsby fundus dystrophy and age-related macular degeneration and suggest a similar phenomenon could be at work in other age-related conditions.