Overall findings

The major contributor of vascular diabetic complications is damage to the ECs [1-22]. Hyperglycemia induced increased oxidative stress causes ECs to secrete large amount of vasoactive factors, growth factors and cytokines [1-22]. These factors lead to structural and functional changes in the ECs such as increased permeability and basement membrane thickening [1-22]. We and others have previously shown that glucose-induced increased oxidative stress alters expression of several genes ultimately causing increased production of ECM proteins by the ECs such as collagen and FN [1-22]. Interestingly some of these above mentioned changes are also seen in normal vascular aging [23-37]. Hence in this study we asked whether hyperglycemia in diabetes accelerates aging-like process in ECs and tissues of diabetic animals (chapter 2).

To create an aging-like in vitro process, we continuously propagated ECs of three different origins (HMEC, BREC and HUVEC) in high and normal glucose and examined for signs of cellular senescence and oxidative stress. We found oxidative stress in hyperglycemia accelerated aging-like changes in ECs. However such processes were variable among the various ECs. Microvascular ECs were more susceptible to glucose induced rapid aging compared to large vessel ECs. As we looked into the mechanism behind such aging process we

found SIRT enzymes were reduced in the aged cells. Glucose-induced increased oxidative stress caused reduction of mitochondrial antioxidant enzymes in a SIRT1 and FOXO1 mediated pathway. Moreover level of SIRT1 was negatively regulated by p300, a HAT and transcriptional co-activator. Using activators of SIRT1 we were able to effectively prevent such aging-like changes in the ECs. In addition, examination of kidneys and retinas of diabetic mice showed that diabetes causes rapid aging in these tissues. Such changes were associated with increased oxidative stress and reduced SIRT1 enzyme levels. Overall we have found an important role played by SIRTs in such process. We showed an important mechanistic pathway of oxidative stress and aging in HG, mediated through FOXO1 and regulated by SIRT1 and p300 (chapter 2).

Next we investigated regulation of SIRT1, specially by miRNAs (chapter 3). miRNAs are post transcriptional regulators of genes. miRNAs are conserved non-coding RNAs and are highly specific in nature, hence holds tremendous potential to be used as therapeutic targets. Several miRNAs have been found to regulate SIRT1 in various physiological and pathological conditions. In this study we examined alteration of SIRT1 targeting miRNAs in the retinal tissues of animals with diabetes.

Our initial microarray analysis showed miR-195, a SIRT1 targeting miRNA was significantly overexpressed in the retinas of diabetic animals. We then validated such findings in two different type of microvascular ECs. This was associated with reduced SIRT1 levels in these cells. We then examined the

possible miR-195 mediated regulation of SIRT1 at multiple levels of complexities in the context of diabetic retinopathy.

We used miR-195 antagomir, mimic and adenovirus with SIRT1 overexpression to identify in vitro biological significance. We found miR-195 antagomir transfection significantly upregulated SIRT1 levels in the ECs. Such transfection further prevented SIRT1 mediated aging changes and reduced glucose-induced increased FN levels in the ECs. SIRT1 overexpression in high glucose showed similar effect whereas miR-195 mimic transfection in NG mimicked the high glucose effect. We also showed similar alterations and its functional significance in an animal model of diabetic retinopathy. By injecting miR-195 antagomir intravitreally in STZ induced type 1 diabetic rats, we showed such treatment prevented SIRT1 reduction in these tissues following 1 month of uncontrolled hyperglycemia. Such treatment also reduced diabetes induced increased vascular permeability and normalized FN proteins levels. Overall in this study we have shown a novel role of miR-195 regulating hyperglycemia induced changes through SIRT1 in microvessels in diabetes. Data from this study indicate that miR-195 may represent a major miRNA, modulating SIRT1 activity in the context of early diabetic microangiopathy.

In order to explore the role of SIRT1 further and to examine whether such mechanisms are universal in other organs affected by diabetic complications, we investigated transgenic mice with SIRT1 overexpression (chapter 4). We induced diabetes in these mice with STZ injection and examined their kidney and retinal

tissues, for possible alteration of genes which are major players of diabetic microangiopathy. We found these transgenic mice had significantly reduced levels of ET-1 and TGF-β1, two major factors of vascular pathogenesis, in the renal and retinal tissues. This finding intrigued us to investigate a possible SIRT1 mediated regulation of ET-1 and TGF-β1 in diabetes.

Using adenoviral overexpression of SIRT1, we found that glucose-induced ET-1 and TGF-β1 upregulation can be markedly reduced by increasing the availability of SIRT1. Such treatment also corrected glucose-induced increased endothelial permeability and increased FN, collagen expressions. Moreover we found such SIRT1 mediated ET-1 and TGF-β1 regulation is mediated through transcriptional co-activator p300. We further found similar relationships in the SIRT1 transgenic mice with diabetes. These findings established that SIRT1 can provide protection against ET-1 and TGF-β1 induced endothelial damage in hyperglycemia. In keeping with our earlier findings we found again that p300 and SIRT1 regulate each other in hyperglycemic conditions and by modulating any of these we can prevent some glucose induced SIRT1 mediated damages.

Together the findings of this research show a novel role played by SIRT1 in diabetic microangiopathy. Data from this study indicate that SIRT1 is a protective molecule in diabetic vascular complications and holds the potential to be used as a therapeutic target for the treatment of diabetes.