Microvessels Of Diabetic Rats Research Articles

P0978THE EFFECTS OF CHRONIC DAPAGLIFLOZIN TREATMENT ON THE RENAL MICROVASCULATURE IN A RAT MODEL OF TYPE 2 DIABETES

Abstract Background and Aims Chronic kidney disease (CKD) is an ever-growing concern and CKD associated with diabetes accounted for ∼ 35% of new cases of end-stage renal failure. Clinical trials with insulin-independent sodium-glucose co-transporter 2 (SGLT2) inhibitors not only showed significant improvements in hyperglycemia and thus renal damage, but also reduced the risk of adverse cardiovascular events. Microvessels supply local tissue oxygen demands and are important controllers of regional perfusion. Endothelial dysfunction has been posited to be an important factor driving the pathogenesis of diabetic nephropathy, largely through impaired eNOS activity and thus reduced nitric oxide bioavailability. Thus, we aim to determine the effects of SGLT2 inhibition on vasodilator function in the renal vasculature. We hypothesize that rats chronically treated with dapagliflozin, a SGLT2 inhibitor, improves renal endothelial function and the microvessels are better able to maintain perfusion in response to the inhibition of nitric oxide synthase and prostaglandin blockade. Method Male Zucker fatty diabetic rats (8 wk old), a model of type 2 diabetes, were given daily oral gavage of 1 mg/kg dapagliflozin or its vehicle for up to 22 wks of age (n=6/group). Renal excretory function, glomerular filtration rate (GFR) and indices of renal injury was assessed before treatment and after every 4 wks up until 22 wks of age. GFR was assessed via transcutaneous clearance of FITC-sinistrin. Vasodilator function of the renal microvasculature was assessed via X-ray microangiography. We conducted successive imaging of the microvessels before and during infusion of acetylcholine (ACh) and sodium nitroprusside (SNP) which facilitated the assessment of endothelium dependent and independent dilation respectively. After which, rats were given successive boluses of L-NAME and indomethacin to assess the vasodilatory function of the microvasculature independent of nitric oxide and prostaglandins respectively. These inhibitors were given under the conditions of SNP clamp which allowed for us to titrate the blood pressure to baseline levels. Lastly, we assessed the ability of endothelium hyperpolarizing factors (EDHFs) to maintain microvascular perfusion when SNP infusion ceased and an infusion of ACh commenced while still under the effects of L-NAME and indomethacin. Results As expected, dapagliflozin alleviated hyperglycemia across the treatment period. There was a tendency for dapagliflozin to ameliorate the decline of GFR, although this apparent effect was not statistically significant. Dapagliflozin did not appear to improve indices of renal injury. Treatment with dapagliflozin alleviated polyuria but did not appear to have an impact on urine osmolarity or sodium excretion. The responses (vessel diameter) of renal microvessels to ACh and SNP was greater in dapagliflozin than in vehicle fed rats. The microvessels of vehicle fed rats appeared to undergo relative constriction in response to L-NAME and indomethacin even under the effects of SNP clamp. In contrast, microvessels of dapagliflozin fed rats appeared to be relatively well-perfused after NOS and COX blockade. This suggests that dapagliflozin may improve endothelial dysfunction commonly associated with diabetic nephropathy. Following NOS and COX blockade, ACh was infused in rats to determine the status of vasodilatory function mediated by EDHFs. The microvessels in diabetic rats did not appear to be dilated after infusion of ACh, suggesting that vasodilatory effects of EDHFs on the vasculature is diminished in diabetic rats. Dapagliflozin appeared to improve this effect in that the renal microvessels were dilated even when NOS and COX production was blocked/inhibited. Conclusion Chronic treatment of dapagliflozin may improve endothelial dysfunction and thus retard the progression of diabetic nephropathy in a rat model of type 2 diabetes.

Increased circulating microparticles in streptozotocin-induced diabetes propagate inflammation contributing to microvascular dysfunction.

Circulating microparticles (MPs) are elevated in many cardiovascular diseases and have been considered as biomarkers of disease prognosis; however, current knowledge of MP functions has been mainly derived from in vitro studies and their precise impact on vascular inflammation and disease progression remains obscure. Using a diabetic rat model, we identified a >130-fold increase in MPs in plasma of diabetic rats compared to normal rats, the majority of which circulated as aggregates, expressing multiple cell markers and largely externalized phosphatidylserine; vascular images illustrate MP biogenesis and their manifestations in microvessels of diabetic rats. Using combined single microvessel perfusion and systemic cross-transfusion approaches, we delineated how diabetic MPs propagate inflammation in the vasculature and transform normal microvessels into an inflammatory phenotype observed in the microvessels of diabetic rats. Our observations derived from animal studies resembling conditions in diabetic patients, providing a mechanistic insight into MP-mediated pathogenesis of diabetes-associated multi-organ microvascular dysfunction. In various cardiovascular diseases, microparticles (MPs), the membrane-derived vesicles released during cell activation, are markedly increased in the circulation. These MPs have been recognized to play diverse roles in the regulation of cellular functions. However, current knowledge of MP function has been largely derived from in vitro studies. The precise impact of disease-induced MPs on vascular inflammation and disease progression remains obscure. In this study we investigated the biogenesis, profile and functional roles of circulating MPs using a streptozotocin-induced diabetic rat model with well-characterized microvascular functions. Our study revealed a >130-fold increase in MPs in the plasma of diabetic rats compared to normal rats. The majority of these MPs originate from platelets, leukocytes and endothelial cells (ECs), and circulate as aggregates. Diabetic MPs show greater externalized phosphatidylserine (PS) than normal MPs. When diabetic plasma or isolated diabetic MPs were perfused into normal microvessels or systemically transfused into normal rats, MPs immediately adhered to endothelium and subsequently mediated leukocyte adhesion. These microvessels then exhibited augmented permeability responses to inflammatory mediators, replicating the microvascular manifestations observed in diabetic rats. These effects were abrogated when MPs were removed from diabetic plasma or when diabetic MPs were pre-coated with a lipid-binding protein, annexinV, suggesting externalized PS to be key in mediating MP interactions with endothelium and leukocytes. Our study demonstrated that the elevated MPs in diabetic plasma are actively involved in the propagation of vascular inflammation through their adhesive surfaces, providing mechanistic insight into the pathogenesis of multi-organ vascular dysfunction that commonly occurs in diabetic patients.

Molecular Imaging of Retinal Endothelial Injury in Diabetic Animals.

Purpose:Diabetic retinopathy is a leading cause of vision loss. There is a great need for early diagnosis prior to the occurrence of irreversible structural damages. Expression of endothelial adhesion molecules is observed before the onset of diabetic vascular damage; however, to date, these molecules cannot be visualized in vivo.Methods:To quantify the expression of endothelial surface molecules, we generated imaging probes that bind to ICAM-1. The α-ICAM-1 probes were characterized via flow cytometry under microfluidic conditions. Probes were systemically injected into normal and diabetic rats, and their adhesion in the retinal microvessels was visualized via confocal scanning laser ophthalmoscopy. Histology was performed to validate in vivo imaging results. Vascular pathologies were visualized using trypsin-digested retinal preparations.Results:The α-ICAM-1 probes showed significantly higher adhesion to retinal microvessels in diabetic rats than in normal controls (P < 0.01), whereas binding of control probes did not differ between the two groups. Western blotting results showed higher ICAM-1 expression in retinas of T1D animals than in normal controls. Retinal endothelial ICAM-1 expression was observed via molecular imaging before markers of structural damage, such as pericyte ghosts and acellular capillaries.Conclusion:Results indicate that molecular imaging can be used to detect subtle changes in the diabetic retina prior to the occurrence of irreversible pathology. Thus, ICAM-1 could serve as a diagnostic target in patients with diabetes. This study provides a proof of principle for non-invasive subclinical diagnosis in experimental diabetic retinopathy. Further development of this technology could improve management of diabetic complications.

Increased Circulating Microparticles in Diabetic Rats are not only a Result of Cell Activation but Actively Serve as Mediators of Inflammatory Signaling

Diabetes is commonly associated with microvascular dysfunction. Our previous study found that microvessels of diabetic rats had increased basal permeability and enhanced permeability responses to inflammatory mediators. We also found that diabetic rats had increased circulating microparticles (MPs). MPs are small vesicles released by activated or apoptotic cells and carry multiple antigens from activated cells. We hypothesized that MPs serve as vectors to actively disseminate inflammation to the whole vascular system. We used flow cytometry to quantify the numbers of MPs and their cell origins. We tested differences between MPs generated by diabetic rats and MPs from normal rats by directly perfusing them into intact microvessels. The effects of MPs on microvessel walls were examined by perfusing matched numbers of isolated Annexin V positive MPs into normal venules for 30 min. After 10 min resumed blood flow in MP perfused vessel, we found significantly higher number of adherent leukocytes in diabetic MP perfused vessels than in normal MP perfused vessels. Diabetic and normal MP‐ mediated leukocyte adhesion were 17 ± 2.9 and 5.7 ± 0.5 leukocytes per 100 μm of vessel length, respectively. Flow cytometry analysis showed that diabetic MPs had an average 7‐fold increase in phosphatidylserine exposure per MP and a 12‐fold increase in platelet‐leukocyte MP aggregates from those in normal plasma MPs. Our results suggest that the population differences and the larger surface area of PS exposed diabetic MPs may contribute to the greater leukocyte adhesion induced by diabetic MPs. These results support our hypothesis that increased MPs in diabetic rats are more than simply biomarkers of the disease but that they are able to directly mediate leukocyte adhesion and actively disseminate inflammation to remote regions.Supported by: 1F32HL114376, P20GM103434, HL56237, HL084338

PAI-1 mRNA expression and myocardial mircovascular change in diabetic rats

Objective To evaluate whether hyperglycemia and ageing influence myocardial microvessels and the expression of myocardial PAI-1 mRNA by observing changes in the number of myocardial microvessels and the expression of myocardial PAI-1 mRNA in aged and young diabetic rats.Methods Aged and young SD rats were randomized to diabetes mellitus(DM) group and control group.The DM model was established by intraperitoneal STZ injection.The rats were raised for 8 weeks and sacrificed.Part of the left ventricular tissue was used for immunohistochemical evaluation,and the remaining part was used for detection of PAI-1 mRNA exprerssion.Myocardial microvascular endothelial specific antigen CD34 was detected by using CD34 antibody and EnVision immunohistochemistry.The number of myocardial microvessels was counted by Medical Image Analysis Software.The expression of myocardial PAI-1 mRNA was detected by real-time fluorescence quantitative PCR.Results The number of myocardial microvessels of DM group was significantly smaller than that of the control in all age groups 8 weeks after modeling(P0.01),and the number of myocardial microvessels of the aged DM group was significantly smaller than that of the young DM group and young control group(P0.01),while there was no significant difference between the aged and young control groups(P0.05).The expression of myocardial PAI-1 mRNA in DM rats of all age groups was significantly increased as compared with the control group at corresponding ages(P0.01).In addition,the expression of myocardial PAI-1 mRNA in the aged control rats was higher than that in the young control rats(P0.05).Conclusion Ageing has a significant influence on the number of myocardial microvessels in diabetic rats but not in non-diabetic rats.Ageing enhances the expression of myocardial AI-1 mRNA in rats.The number of myocardial microvessels is decreased and the expression of myocardial PAI-1 mRNA is enhanced in diabetic rats at all ages.

A PKC-β inhibitor protects against cardiac microvascular ischemia reperfusion injury in diabetic rats

PKC-beta inhibitor Ruboxistaurin (RBX or LY333531) can be used to reverse diabetic microvascular complication. However, it has not been previously established whether RBX can protect against ischemia/reperfusion (I/R) injury of cardiac microvessels in diabetic rats. STZ-induced diabetic rats were randomized into four groups and underwent I/R procedures. Cardiac barrier function and the region of cardiac microvascular lesion were examined. Cell monolayer barrier function was detected in cultured cardiac microvascular endothelial cells (CMECs) subjected to simulated I/R (SI/R). PKC-beta siRNA was transfected into CMECs to silence PKC-beta. Apoptosis Index of CMECs was detected by TUNEL assay and phosphor-LIMK2 protein expression was examined by Western blot analysis. RBX and insulin administration significantly reduced the cardiac microvascular lesion region and Apoptosis Index of endothelial cells (all P < 0.05 vs. no-treatment group). RBX decreased phosphor-LIMK2 expression (P < 0.05 vs. no-treatment group). RBX pretreatment and transfection with PKC-beta siRNA induced a rapid barrier enhancement in CMECs monolayer as detected by increased transendothelial electrical resistance (TER) and decreased FITC-dextran clearance (all P < 0.05 vs. no-treatment group). Meanwhile, RBX pretreatment and transfection with PKC-beta siRNA significantly decreased TUNEL positive CMECs and phosphor-LIMK2 expression in cultured CMECs (all P < 0.05 vs. no-treatment group). RBX pretreatment reduced F-actin/G-actin in cultured CMECs, reproducing the same effect as PKC-beta siRNA. These data indicate that PKC-beta inhibitor (RBX) may be helpful in attenuating the risk of severe cardiac microvascular I/R injury in diabetic rats partly due to its maintenance of endothelial barrier function and anti-apoptotic effect.

Retinal Microvascular Patency in the Diabetic Rat

To study whether the patency to erythrocytes in retinal microvessels of diabetic rats is reduced or blocked before the vessels lose their patency to plasma flow. We used recognized techniques to induce diabetic and galactose related microvascular retinal lesions in rats: (1) alloxan induction (2) streptozotocin induction (3) galactose-containing diet. The rats were followed up to 17 months. We used our vascular trichrome technique to observe the effects of the ongoing diabetes on the retinal microcirculation. A focal leakage of a plasma-borne fluorescent dye was noted around the junction of the deep retinal capillaries and the ascending venules to the superficial retinal circulation in the streptozotocin and alloxan diabetic rats by the 14th month, and, by the 16th month, retinal capillary non-perfusion and retinal vascular malformations were present. The affected vessels showed patency to microspheres (0.2 microm in diameter) but no perfusion of erythrocytes. No such changes were seen in the galactose-fed rats. (1) The location between the deep retinal capillary net and the ascending venules may be the site of early vascular leakage in the diabetic rat model, (2) the erythrocytes' passage in the affected retinal microcirculation was blocked before the development of complete blockage to plasma in diabetic rats. The logical assumption that during the development stage of retinal capillary occlusion there may be a transient stage of microvascular insufficiency was examined. The lathyrogen, imino-diproprionitrile (IDPN), had previously been effective for creating a fast-developing model of retinal vasculopathy. Using that model, we demonstrated a stage in which the retinal microvasculature was blocked to erythrocytes but not to plasma [1]. However, we questioned the applicability of our findings to more slowly developing microvasculopathies, such as diabetic retinopathy. We designed the current study to examine the presence of such stage in slowly developing microvasculopathy. Animal models that are known to induce "diabetic retinopathy-like" changes used [2-4]. The diabetic animals were followed for a period of 17 months. Starting at the 12th month, a few animals of each group were killed and the retains were examined with our trichrome method [1] for relative capillary patency to erythrocytes and plasma, for functionality of endothelial cells, and for disturbances in the blood-retinal barrier. The results of this study support the hypothesis that retinal microvascular insufficiency does exist as a temporary stage that precedes the development of complete capillary blockage in long-term developing rat models of diabetic retinopathy.

Effects of cilostazol on the peripheral nerve function and structure in STZ-induced diabetic rats

We examined the effect of cilostazol (CZ), antiplatelet agent and potent vasoactive compound, which has an inhibitory effect on tissue phosphodiesterase, on peripheral nerve in streptozotocin-induced diabetic rats. Diabetic rats were fed for 12 weeks with a chow containing 0.01% or 0.03% CZ ( w w ) and the results were compared with untreated diabetic rats. The 0.03% CZ treatment significantly improved motor nerve-conduction velocity and restored nerve Na +, K +-ATPase activity in diabetic rats without affecting body weight and glycated hemoglobin levels, but the effects of 0.01% CZ treatment did not reach statistical difference. Elevated sorbitol and reduced myo-inositol levels in diabetic nerve tissues were not influenced by CZ treatment. Structural analysis of the sural nerve demonstrated a partial but significant effect on decreased mean myelinated fiber area and atrophic changes of the axon in diabetic rats treated with 0.01% CZ. CZ treatment inhibited reduction of pericyte area of endoneurial microvessels in diabetic rats. Expansion of endoneurial microvessels and luminal area in relation to vascular area also tended to be inhibited by CZ treatment. Thus CZ treatment ameliorated, although not completely, functional and structural abnormalities in peripheral nerve of diabetic rats without effecting the polyol pathway. These results support the contention that vascular factors may play an important role in the etiology of experimental diabetic neuropathy and suggest that CZ may have a beneficial therapeutic effect on diabetic neuropathy.

Effects of Aminoguanidine on Structural Alterations of Microvessels in Peripheral Nerve of Streptozotocin Diabetic Rats

This study was designed to explore the effect of aminoguanidine (AG) on the structural abnormalities of microvessels in the sural nerve of streptozotocin-induced diabetic rats. Diabetic rats were treated with AG (25 mg/kg, sc) for 16 weeks, a dose high enough to prevent glycation but also inhibit inducible nitric oxide synthase. Nontreated diabetic rats and normal Wistar rats were used for comparison. Morphometric analysis disclosed an expansion of vascular and luminal areas of endoneurial microvessels in diabetic rats, particularly of large size, compared to control rats. These changes were partially but significantly corrected by AG treatment. There was no closure of microvessels in diabetic rats. The mean ratio of basement membrane area to vascular area was significantly smaller in diabetic rats than that in normal control rats and basement membrane was not thickened in diabetic rats. The mean ratio of endothelial cell area to vascular area was smaller in diabetic rats than that in nondiabetic control rats, and it was not influenced by AG treatment. Numbers of microvessels per unit endoneurial area in diabetic rats were not different from those of control rats. These findings in conjunction with previous reports suggest that alterations of nerve blood flow detected in diabetic peripheral nerve may be relevant to structural alterations of endoneurial microvessels, which may be an early sign of endoneurial microangiopathy. We also consider that AG treatment is beneficial for experimental diabetic neuropathy, partially through the correction of endoneurial microcirculation.

Diabetes-induced alterations of glucose metabolism in rat cerebral microvessels.

The effect of diabetes on the metabolism of glucose and lactate was examined in isolated rat cerebral microvessels. In rats with diabetes induced with streptozotocin, glucose oxidation to CO2 by the microvessels was decreased by 54-83% and its conversion to lactate by 21-61%. Insulin therapy for several days or starvation for 48 h both lowered blood glucose levels in the diabetic rats and restored microvessel glucose metabolism to normal. Cerebral microvessels consist principally of the capillaries that constitute the blood-brain barrier. Direct assessment of the blood-brain barrier in vivo using the brain uptake index (BUI) technique revealed a close parallel to the findings in the microvessels. Thus, hexose transport was diminished in diabetic rats and restored to normal by both insulin therapy and starvation. The oxidation of [1-14C]lactate to CO2 like that of glucose was depressed in microvessels of diabetic rats. In contrast to glucose, however, the transport of lactate across the blood-brain barrier in vivo was not altered. These findings suggest that diabetes suppresses glucose metabolism in rat cerebral microvessels and downregulates glucose transport across the blood-brain barrier. They also suggest that both of these processes are regulated by chronic alterations in blood glucose concentration rather than by insulin per se.