الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Diabetes remains a main risk factor for CV disorders.The vascular impact of diabetes is typically related to poor glycemic control. Yet, a significant proportion of diabetic patients present with microvascular complications at initial diagnosis, implicating a vascular insult early in the course ofmetabolic dysfunction.
On the other hand, recent evidence suggests that some anti-diabetic drugsmight have a positive impact on diabetic cardiovascular complications apart from their hypoglycemic effect.
The mechanism of the early insult occurring in thecontext of diabetes development is not well-defined.
Furthermore, the potential corrective mechanism of anti-diabetic drugs was not properly investigated in these circumstances.
In the context of the current study, we used a rat model of 12-week mild hypercaloric intake to study the mechanism of early vascular dysfunction in the course of development of diabetes.
We investigated the metabolic effect of MHC diet, the outcome on the structure and function of blood vessels, the specific mechanisms implicatedin vascular dysfunction induced by MHC diet, the incidence of perivascular adipose inflammation before the onset of diabetes, and the potential of some anti-hyperglycemic drugs (specifically metformin and pioglitazone) in modulating the impaired vascular functions in MHC model independent of their hypoglycemic effect.Asummary of the main results and conclusions of the current study is outlined below.1.Our rat model of hypercaloric intake was employed to represent mild metabolic challenge.
The mild hypercaloric (MHC) model receives nearly 38% of energy intake as fat, which is slightly higher than the ADA guidelines for recommended daily fat intake (20-35% energy required as fat), while still within the range of compositions of high-fat diet (which is 20% to60% energy required as fat) previously well-documented and employed.
A diet composition including fructose was selected to accurately simulate Western diets enriched in refined sugars in addition to saturated fat formerly associated with CV abnormalities in humans and rats.2.Our current results showed that this diet resulted in stable fasting hyperglycemia after 16 weeks of feeding i.e.development of diabetes, however, after 12 weeks of MHC diet, there were no significant differences between control group and MHC-fed group in body weight, FBG levels, and OGT.
This offers the advantage to investigate any structural, functional or molecular aberrations appearing in the early stage of metabolic challenge apart from hyperglycemia or obesity interference. Yet at this stage,elevated serum levels of insulin, total cholesterol, TGs, and non-HDL cholesterol were observed as the MHC diet-induced metabolic insult.
3.Upon evaluating vascular structure and functions of MHC aortic tissues, many abnormalities were observed such as elevated sensitivity to PE-induced vasoconstriction in aortic rings. There was no significant difference in the PE-induced contraction between intact and denuded aortic rings in either control or MHC groupsindicating that the observed difference was due to an alteration in smooth muscle contractile function.
<Rats consuming MHC diet had a significantly higher increase in mean arterial pressure (MAP) in response to PE administered intravenously, especially at higher doses compared to control ones.
Additionally, third order mesenteric arterioles from MHC rats studied as a representative vessel of the resistance circulation showed greater constriction to PE treatment compared to control ones.
Summary and Conclusions79794.PE concentration response curves were studied in buffer solutions with or without the physiological calcium concentration. Interestingly, calcium-free Krebs did not affect the PE-induced contraction of aortic rings taken from MHC rats, while reducing the maximum contraction induced by PE in aortic rings of control rats by ~50%, indicating the possibility of recruiting calcium-independent mechanisms of contraction in MHC rats. While H1152 reducedthe PE-induced contraction by 50% in control aortic rings, there was no significant reduction in the maximal contraction of aortic rings from MHC rats, indicating a potential basal increase in ROCK-dependent calcium sensitization.
This difference was not observed upon treatment with 3 M of the PKC inhibitor GF109203X. GF109203X reduced the maximal contractile tone equally in aortic rings obtained from both control and MHC rats indicating that the observed defect was only in ROCK-mediated calcium sensitization.
5.Immunohistochemical staining and western blotting showed that there was no significant difference in the expression of ROCK-1 in aortic smooth muscle tissue between MHC rats and control ones.
Phosphorylation of MYPT1 at T855 was measured as an indicator of ROCK activity inducing calcium sensitization.
Interestingly, control rats showed minimal phosphorylation of MYPT at T855 at basal conditions without further decrease occurring in presence of H1152.
<While in the presence of PE, phosphorylation of MYPT increased significantly as expected. On the other hand, MHC rats showed elevated basal phosphorylation levels of MYPT at T855 that were decreased in presence of H1152, indicating an elevated basal calcium sensitization potentially leading to an enhanced contractile response to PE. No further increase in MYPT phosphorylation was observed in MHC tissues upon treatment with PE.
<Thus, the basal activity of ROCK-1, rather than its expression, is elevated in aortic smooth muscles from MHC-fed rats possibly owing to inflammation.6.PE-induced maximal contraction of control aortic rings was significantly reduced either in the presence of metformin (10 μM) or pioglitazone (10 μM) compared to untreated time-matched controls.
Similarly, PE-induced maximal contraction of MHC rings was significantly reduced either in the presence of metformin or pioglitazone, indicating a potential direct effect on vascular contractility in both control and pathological states.
This could possibly be attributed to the observed acute elevation of AMPK phosphorylation. Both pioglitazoneand metformin increased the levels of phosphorylated AMPK acutely compared to control.
7.Following two-week treatment of MHC rats with pioglitazone and metformin, PE concentration-response curves exhibited a rightward shift (~ 4-fold increase in EC50for pioglitazone, P< 0.05, and ~2-fold increase in EC50for metformin, P<0.05) and maximal contraction was significantly reduced compared to untreated MHC rats, indicating the ability of pioglitazone and metformin to ameliorate the vascular abnormalities elicited by MHC diet. Similar to control rats, 0.5 M of H1152 led to a significant reduction of the maximal contraction of aortic rings from both metformin-treated and pioglitazone-treated MHC-fed rats, signifying the reversal of the abnormal increase in basal ROCK activity triggered in MHC-fed rats. PKC inhibition by 3 M GF109203X reduced the maximal contraction in both metformin and pioglitazone –treated rats similar to control group, indicating that both metformin and pioglitazone do not affect PKC.No further reduction of the basal MYPT1 phosphorylation was detected upon treatment of aortic rings from metformin-and pioglitazone-treated rats by H1152.
<Summary and Conclusions80808.In order to investigate whether the functional defect triggered by MHC feeding was associated with inflammatory changes leading to structural and signaling abnormalities, serialsections and protein extracts of thoracic aorta from control and different treated rat groups were subjected to microscopical and western blotting examination of different markers. H&E staining showed an increased medial thickness in sections from MHC fed rats compared to control.
Either metformin or pioglitazone treatment reversed the increased medial thickness to values similar to the control. Immunostaining for TGF-β1 and Smad3 showed an increased expression of both proteins in MHC-fed rats, which was normalized upon treatment with metformin and pioglitazone. DHE staining for ROS showed an increase in aortic tissues from MHC-fed rats and a parallel reduction in those from rats treated with metformin and pioglitazone. Additionally, phosphorylated AMPK was suppressed in aortic tissues from MHC rats compared to controls, and was restored in tissues from MHC rats treated with either metformin or pioglitazone.
Phosphorylated Erk1/2 was significantly higher in MHC rats compared to control ones, and returned to control levels in MHC rats treated with either metformin or pioglitazone.
Protein expression levels of the pro-inflammatory cytokine, interleukin-1β were significantly higher in MHC rats compared to controls. This increased expression was abolished in tissues from MHC rats treated with either metformin or pioglitazone.9.Markers of systemic inflammation were measured in the serum.
After 12 weeks of feeding, levels of advanced glycation end products in the serum of MHC rats were not significantly different compared to control group, indicating that MHC diet does not generate AGEs during this period. Levels of TGF-β1 and IL-1β in the serum of MHC rats were not significantly different compared to control group ruling out a role for systemic inflammation in the observed vascular dysfunction.
To confirm this observation, expression of both TGF-β1 and IL-1β in visceral adipose, as a representative of a systemic adipose pool, was examined by quantitative PCR. No significant increase in the transcripts of either cytokine was detected in this tissue. This indicated that 12-week MHC diet is not associated with systemic inflammation.10.MHC diet induced localized inflammation in the PVAT.
An increased level of different inflammatory markers was observed upon examination of perivascular adipose tissue obtained from thoracic aorta. Evidence of adipose tissue hypoxia was apparent in the increased expression of HIF-1α in MHC rats compared to control rats. Consequent inflammatory changes were detected as an increased expression of NF-κB and a reduced expression of PPAR-γ.
Immunohistochemical examination confirmed the inflammatory phenotype in perivascular adipose tissue. CD68 immunostaining demonstrated an increased crown-like structures (macrophages surrounding adipocytes) in perivascular adipose tissue compared from MHC-fed rats to controls, in addition to adipocyte hypertrophy as evident by Oil Red Staining. In parallel, mRNA levels of both inflammatory cytokines, IL-1β and TGF-β, were increased in perivascular adipose tissue from MHC-fed rats. Significantly, apart from PPAR-γ expression levels, a 2-week treatment with either metformin or pioglitazone reversed increased inflammatory marker expression without affecting blood glucose levels coincident with the amelioration of the vascular functional, structural and inflammatory changes at this stage during the development of metabolic disease.
Unlike pioglitazone, the effect of metformin was not associated with a large reduction in adipocyte size and serum insulin levels indicating a potentially direct anti-inflammatory effect.
<Both pioglitazone and metformin normalized.
Summary and Conclusions8181the expression of inflammatory markers in PVAT due to MHC diet such asHIF-1α, NF-κB, ROS, IL-1β and TGF-β.11.The effect of reduction of the caloric load on aortic structure and function in MHC-fed rats was examined in a rat group switched to normal chow for the last two weeks of feeding. Importantly, PE concentration response curve of aortic rings from MHC-NC group exhibited a rightward shift compared to MHC group (~3-fold increase in EC50, P<0.05), along with lower maximal contraction.
Additionally, switching to normal chow normalized the response to H1152 and basal ROCK activity as indicated by MYPT1 phosphorylation. Moreover, aortic tissues from MHC-fed rats switched to normal chow showed normal intimal thickness and reduced levels of ROS, and TGF-β1 and interleukin-1β expression similar to the control.
There were no changes in AMPK and Erk1/2 phosphorylation levels compared to control rats.12.ACh-mediated endothelium-dependent relaxations of aortic rings from MHC rats were impaired compared to those taken from control rats.
Not only did these rings showreduced relaxations, they also tended to show a reversal of the response towards constriction at higher ACh concentrations.
An equivalent dilatory response in rings from control and MHC-fed rats to SNP indicated that this aberration in endothelium-dependent vasodilation is probably due to an endothelial deficit rather than a reduced ability of vascular smooth muscle to relax.
<No role for increased production of endothelin, 20-HETE, or ROS produced by NOS uncoupling was detected.13.A residualrelaxation of over 25% in response to ACh persisted in presence of L-NAME and indomethacin, indicating a potential contribution from a third endothelium-derived relaxing agent other than NO and prostanoids.
As such, the role of endothelium-derived hyperpolarization was examined. This role was confirmed by the use of barium chloride to blockKir channels and interfere with endothelium-dependent hyperpolarization.
Interestingly, blockade of Kir channels in aortic rings from control rats produced an ACh response profile similar to that of MHC-fed rats with a tendency for terminal constriction. On the other hand, no role for endothelium-dependent hyperpolarization was observed in aortic rings from MHC-fed rats.
< This was further confirmed by a reduction of thedilatory response to diazoxide, a potassium channel opener, and Kir 2.1 channel expression in aortic tissue from MHC-fed rats.14.MHC rats treated with either metformin or pioglitazone exhibited an improvement in the response to the NO component of endothelium-dependent vasodilation apparent as an increased sensitivity to treatment with L-NAME.
This was also reflected in the improvement of endothelial-dependent relaxation in response to insulin.
However, neither metformin nor pioglitazone treatment led to an enhancement of the impaired endothelium dependent hyperpolarization in aortic rings from MHC rats.
<This was observed as a lack of effect of barium chloride on the ACh-mediated response in either condition. Furthermore, neither drug treatment was able to improve the reduced dilatory response to diazoxide in aortic rings from MHC-fed rats.
<This is in line with the finding that serum cholesterol levels remained high in MHC rats treated with either metformin or pioglitazone.15.In conclusion, the results of the present study provide a framework for the potential mechanisms underlying early vascular dysfunction in the course of diabetes development.
<Indeed, vascular structural and functional abnormalities were observed well beforethe development of hyperglycemia emphasizing the importance of early intervention with vascular complications. Our results also highlight the role of Summary and Conclusions8282potential pleiotropic effects of anti-diabetic drugs in vasculoprotection.
Additional detailed mechanistic and clinical studies are required in order to enable tailoring of anti-diabetic therapy and general vasculoprotective therapy in metabolic disease to minimize the incidence and progression of vascular complications.
As well, therapeutic interventions targeting adipose inflammation might be necessary to prevent vascular complications in euglycemic patients.
Future research is required to elucidate the changes in signaling cascades that accompany the pathophysiological transformation in vascular function from the prediabetic to the diabetic stages