Gellan gum-hyaluronate spongy-like hydrogels promote angiogenesis in hindlimb ischemia

"Tissue engineering: part A", vol. 21, suppl. 1 (2015)

Tramadol Alleviates Myocardial Injury Induced by Acute Hindlimb Ischemia Reperfusion in Rats

AbstractBackground:Organ injury occurs not only during periods of ischemia but also during reperfusion. It is known that ischemia reperfusion (IR) causes both remote organ and local injuries.Objective:This study evaluated the effects of tramadol on the heart as a remote organ after acute hindlimb IR.Methods:Thirty healthy mature male Wistar rats were allocated randomly into three groups: Group I (sham), Group II (IR), and Group III (IR + tramadol). Ischemia was induced in anesthetized rats by left femoral artery clamping for 3 h, followed by 3 h of reperfusion. Tramadol (20 mg/kg, intravenous) was administered immediately prior to reperfusion. At the end of the reperfusion, animals were euthanized, and hearts were harvested for histological and biochemical examination.Results:The levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were higher in Groups I and III than those in Group II (p < 0.05). In comparison with other groups, tissue malondialdehyde (MDA) levels in Group II were significantly increased (p < 0.05), and this increase was prevented by tramadol. Histopathological changes, including microscopic bleeding, edema, neutrophil infiltration, and necrosis, were scored. The total injuryscore in Group III was significantly decreased (p < 0.05) compared with Group II.Conclusion:From the histological and biochemical perspectives, treatment with tramadol alleviated the myocardial injuries induced by skeletal muscle IR in this experimental model.

Effect of NF kappa B Inhibition by CAPE on Skeletal Muscle Ischemia-Reperfusion Injury

Background/Aims. Nuclear factor kappa B (NF kappa B) plays important role in the pathogenesis of skeletal muscle ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent NF kappa B inhibitor, exhibits protective effects on I/R injury in some tissues. In this report, the effect of CAPE on skeletal muscle I/R injury in rats was studied. Methods. Wistar rats were submitted to sham operation, 120-min hindlimb ischemia, or 120-min hindlimb ischemia plus saline or CAPE treatment followed by 4-h reperfusion. Gastrocnemius muscle injury was evaluated by serum aminotransferase levels, muscle edema, tissue glutathione and malondialdehyde measurement, and scoring of histological damage. Apoptotic nuclei were determined by a terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay. Muscle neutrophil and mast cell accumulation were also assessed. Lipoperoxidation products and NF kappa B were evaluated by 4-hydroxynonenal and NF kappa B p65 immunohistochemistry, respectively. Results. Animals submitted to ischemia showed a marked increase in aminotransferases after reperfusion, but with lower levels in the CAPE group. Tissue glutathione levels declined gradually during ischemia to reperfusion, and were partially recovered with CAPE treatment. The histological damage score, muscle edema percentage, tissue malondialdehyde content, apoptosis index, and neutrophil and mast cell infiltration, as well as 4-hydroxynonenal and NF kappa B p65 labeling, were higher in animals submitted to I/R compared with the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. Conclusions. CAPE was able to protect skeletal muscle against I/R, injury in rats. This effect may be associated with the inhibition of the NF kappa B signaling pathway and decrease of the tissue inflammatory response following skeletal muscle I/R. (C) 2009 Elsevier Inc. All rights reserved.

Putative role of ischemic postconditioning in a rat model of limb ischemia and reperfusion: involvement of hypoxia-inducible factor-1α expression

Hypoxia-inducible factor-1α (HIF-1α) is one of the most potent angiogenic growth factors. It improves angiogenesis and tissue perfusion in ischemic skeletal muscle. In the present study, we tested the hypothesis that ischemic postconditioning is effective for salvaging ischemic skeletal muscle resulting from limb ischemia-reperfusion injury, and that the mechanism involves expression of HIF-1α. Wistar rats were randomly divided into three groups (n=36 each): sham-operated (group S), hindlimb ischemia-reperfusion (group IR), and ischemic postconditioning (group IPO). Each group was divided into subgroups (n=6) according to reperfusion time: immediate (0 h, T0), 1 h (T1), 3 h (T3), 6 h (T6), 12 h (T12), and 24 h (T24). In the IPO group, three cycles of 30-s reperfusion and 30-s femoral aortic reocclusion were carried out before reperfusion. At all reperfusion times (T0-T24), serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities, as well as interleukin (IL)-6, IL-10, and tumor necrosis factor-α (TNF-α) concentrations, were measured in rats after they were killed. Histological and immunohistochemical methods were used to assess the skeletal muscle damage and HIF-1α expression in skeletal muscle ischemia. In groups IR and IPO, serum LDH and CK activities and TNF-α, IL-6, and IL-10 concentrations were all significantly increased compared to group S, and HIF-1α expression was up-regulated (P<0.05 or P<0.01). In group IPO, serum LDH and CK activities and TNF-α and IL-6 concentrations were significantly decreased, IL-10 concentration was increased, HlF-1α expression was down-regulated (P<0.05 or P<0.01), and the pathological changes were reduced compared to group IR. The present study suggests that ischemic postconditioning can reduce skeletal muscle damage caused by limb ischemia-reperfusion and that its mechanisms may be related to the involvement of HlF-1α in the limb ischemia-reperfusion injury-triggered inflammatory response.

Effect of cilostazol and pentoxifylline on gait biomechanics in rats with ischemic left hindlimb

Objective: The purpose of this study was to determine the impact of pharmacologic treatment with cilostazol and pentoxifylline on gait biomechanics of ischemic rat hindlimbs compared with nonischemic controls. Methods: An experimental study was designed using 30 Wistar rats divided into five groups (n = 6): control (C); ischemia (I) - animals submitted to left common iliac artery interruption without pharmacologic treatment; pentoxifylline (Pen) - rats submitted to procedure and treated with pentoxifylline 3 mg/kg twice a day for 6 weeks; cilostazol (Cil) - animals submitted to procedure and treated with cilostazol 30 mg/kg twice a day for 6 weeks; and sham (S) - animals submitted to procedure without artery interruption. Gait analysis was performed using a computed treadmill. Time, number, and duration of each hindlimb contact were obtained. The total number of contacts (TNC) and the total duration of contacts (TDC) were compared between left and right hindlimb and among groups. Left hindlimb ischemic incapacitation index (LHII) was defined by the formula: LHII = (1 - TNCleft x TDCleft/TNCright x TDCright) x 100 Results: Left hindlimb TNC values were twofold lower in I, Pen, and Cil groups than in C and S groups (P < .01). In I, Pen, and Cil groups, TNC values for the left hindlimb were half of the right hindlimb ones (P < .01). Left hindlimb TDC values were lower in I and Pen groups than the other groups (P < .01). Cil group presented twofold increased values, not different from C and S groups (P = 0.16). Right hindlimb TNC values were greater for I group (P < .01). LHII was around zero in C and S groups and 82 in both I and Pen groups (P < .01). Cil group presented a LHII of 42; higher than C and S groups, but lower than I and Pen groups (P < .01). Conclusions: Cilostazol at a dose of 30 mg/kg twice a day promoted improvement in gait performance in rats submitted to chronic hindlimb ischemia. Pentoxifylline at a dose of 3 mg/kg twice a day did not show this effect. (J Vasc Surg 2012;56:476-81.)

Stromal vascular fraction cell sheets angiogenic potential for tissue engineering and regenerative medicine applications

One of the biggest concerns in the Tissue Engineering field is the correct vascularization of engineered constructs. Strategies involving the use of endothelial cells are promising but adequate cell sourcing and neo-vessels stability are enduring challenges. In this work, we propose the hypoxic pre-conditioning of the stromal vascular fraction (SVF) of human adipose tissue to obtain highly angiogenic cell sheets (CS). For that, SVF was isolated after enzymatic dissociation of adipose tissue and cultured until CS formation in normoxic (pO2=21%) and hypoxic (pO2=5%) conditions for 5 and 8 days, in basal medium. Immunocytochemistry against CD31 and CD146 revealed the presence of highly branched capillary-like structures, which were far more complex for hypoxia. ELISA quantification showed increased VEGF and TIMP-1 secretion in hypoxia for 8 days of culture. In a Matrigel assay, the formation of capillary-like structures by endothelial cells was more prominent when cultured in conditioned medium recovered from the cultures in hypoxia. The same conditioned medium increased the migration of adipose stromal cells in a scratch assay, when compared with the medium from normoxia. Histological analysis after implantation of 8 days normoxic- and hypoxic-conditioned SVF CS in a hindlimb ischemia murine model showed improved formation of neo-blood vessels. Furthermore, Laser Doppler results demonstrated that the blood perfusion of the injured limb after 30 days was enhanced for the hypoxic CS group. Overall, these results suggest that SVF CS created under hypoxia can be used as functional vascularization units for tissue engineering and regenerative medicine.

Experimental peripheral arterial disease: new insights into muscle glucose uptake, macrophage, and T-cell polarization during early and late stages.

Peripheral arterial disease (PAD) is a common disease with increasing prevalence, presenting with impaired walking ability affecting patient's quality of life. PAD epidemiology is known, however, mechanisms underlying functional muscle impairment remain unclear. Using a mouse PAD model, aim of this study was to assess muscle adaptive responses during early (1 week) and late (5 weeks) disease stages. Unilateral hindlimb ischemia was induced in ApoE(-/-) mice by iliac artery ligation. Ischemic limb perfusion and oxygenation (Laser Doppler imaging, transcutaneous oxygen pressure assessments) significantly decreased during early and late stage compared to pre-ischemia, however, values were significantly higher during late versus early phase. Number of arterioles and arteriogenesis-linked gene expression increased at later stage. Walking ability, evaluated by forced and voluntary walking tests, remained significantly decreased both at early and late phase without any significant improvement. Muscle glucose uptake ([18F]fluorodeoxyglucose positron emission tomography) significantly increased during early ischemia decreasing at later stage. Gene expression analysis showed significant shift in muscle M1/M2 macrophages and Th1/Th2 T cells balance toward pro-inflammatory phenotype during early ischemia; later, inflammatory state returned to neutrality. Muscular M1/M2 shift inhibition by a statin prevented impaired walking ability in early ischemia. High-energy phosphate metabolism remained unchanged (31-Phosphorus magnetic resonance spectroscopy). Results show that rapid transient muscular inflammation contributes to impaired walking capacity while increased glucose uptake may be a compensatory mechanisms preserving immediate limb viability during early ischemia in a mouse PAD model. With time, increased ischemic limb perfusion and oxygenation assure muscle viability although not sufficiently to improve walking impairment. Subsequent decreased muscle glucose uptake may partly contribute to chronic walking impairment. Early inflammation inhibition and/or late muscle glucose impairment prevention are promising strategies for PAD management.

Neovascularization after ischemic injury: evaluation with 99mTc-HYNIC-RGD

Purpose: Angiogenesis involves many mediators including integrins, and the tripeptide RGD is a target amino acid recognition sequence for many of them. Hindlimb ischemia is a simple and convenient animal model however standardization of the injection procedures in the devascularized and control limb is lacking, thus rendering difficult the interpretation of results. The aim of this investigations was to evaluate neovascularization in a hindlimb murine model by means of 99mTc-HYNIC-ß-Ala-RGD. Methods: 99mTc-HYNIC-RGD analog was prepared using coligands. Ischemia was induced in Wistar rats by double- ligation of the common femoral artery. Radiolabeled RGD was injected after 2h, as well as 1, 3, 5, 7, 10 and 14 days. Uptake was evaluated by planar imaging and biodistribution studies. Results: The highest ratio between ischemia and control was achieved at the 7th day (2.62 ± 0.95), with substantial decrease by the 14th day. For pertechnetate the 7th day ratio was 0.87 ± 0.23. Scintigraphic image confirmed different uptakes. Conclusion: 99mTc-HYNIC-RGD analog concentrated in ischemic tissue by the time of widespread angiogenesis and pertechnetate confirmed reduction in blood flow. In this sense, the protocol can be recommended for ischemic models.

Novel cell-free strategy for therapeutic angiogenesis: in vitro generated conditioned medium can replace progenitor cell transplantation

BACKGROUND: Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy. METHODOLOGY/PRINCIPAL FINDINGS: EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2+/-2.9% and 83.7+/-3.0% vs. 53.5+/-2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62+/-0.03 and 1.68+/-0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6+/-0.3 and 8.1+/-0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7+/-44.1 vs. 340.0+/-29.1 CD34(+)/CD45(-) cells/1x10(5) mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9+/-0.7 vs. 2.6+/-0.4 CD34(+) cells/HPF, P<0.001) 3 days after the last injection. CONCLUSIONS/SIGNIFICANCE: Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.

Hind limb ischemia in rabbit model: T2-prepared versus time-of-flight MR angiography at 3 T.

PURPOSE: To prospectively compare various parameters of vessels imaged at 3 T by using time-of-flight (TOF) and T2-prepared magnetic resonance (MR) angiography in a rabbit model of hind limb ischemia. MATERIALS AND METHODS: Experiments were approved by the institutional animal care and use committee. Endovascular occlusion of the left superficial femoral artery was induced in 14 New Zealand white rabbits. After 2 weeks, MR angiography and conventional (x-ray) angiography were performed. Vessel sharpness was evaluated visually in the ischemic and nonischemic limbs, and the presence of small collateral vessels was evaluated in the ischemic limbs. Vessel sharpness was also quantified by evaluating the magnitude of signal intensity change at the vessel borders. RESULTS: The sharpness of vessels in the nonischemic limbs was similar between the TOF and the T2-prepared images. In the ischemic limbs, however, T2-prepared imaging, as compared with TOF imaging, generated higher vessel sharpness in arteries with diminished blood flow (mean vessel sharpness: 44% vs 30% for popliteal arteries, 45% vs 28% for saphenous arteries; P < .001 for both comparisons) and enabled better detection of small collateral vessels (93% vs 36% of vessels, P < .001). CONCLUSION: T2-prepared imaging can facilitate high-spatial-resolution MR angiography of small vessels with low blood flow and thus has potential as a tool for noninvasive evaluation of arteriogenic therapies, without use of contrast material. Supplemental material: http://radiology.rsnajnls.org/cgi/content/full/2452062067/DC1.

Gut Bacteria Products Prevent Aki Induced By Ischemia-reperfusion.

Short-chain fatty acids (SCFAs) are fermentation end products produced by the intestinal microbiota and have anti-inflammatory and histone deacetylase-inhibiting properties. Recently, a dual relationship between the intestine and kidneys has been unraveled. Therefore, we evaluated the role of SCFA in an AKI model in which the inflammatory process has a detrimental role. We observed that therapy with the three main SCFAs (acetate, propionate, and butyrate) improved renal dysfunction caused by injury. This protection was associated with low levels of local and systemic inflammation, oxidative cellular stress, cell infiltration/activation, and apoptosis. However, it was also associated with an increase in autophagy. Moreover, SCFAs inhibited histone deacetylase activity and modulated the expression levels of enzymes involved in chromatin modification. In vitro analyses showed that SCFAs modulated the inflammatory process, decreasing the maturation of dendritic cells and inhibiting the capacity of these cells to induce CD4(+) and CD8(+) T cell proliferation. Furthermore, SCFAs ameliorated the effects of hypoxia in kidney epithelial cells by improving mitochondrial biogenesis. Notably, mice treated with acetate-producing bacteria also had better outcomes after AKI. Thus, we demonstrate that SCFAs improve organ function and viability after an injury through modulation of the inflammatory process, most likely via epigenetic modification.

Predictors Of Silent Myocardial Ischemia In Resistant Hypertensive Patients.

Hypertension is the most prevalent and significant modifiable risk factor for coronary heart disease. A portion of patients with uncontrolled hypertension are considered to have resistant hypertension (RHTN). Myocardial ischemia incidence increases along with blood pressure (BP) levels. However, the prevalence of myocardial ischemia in patients with RHTN, as well as the factors associated with it, is unknown. We enrolled 129 patients with true RHTN regularly followed in our specialty hypertension clinic and evaluated then by resting and dipyridamole pharmacological stress myocardial perfusion scintigraphy. Patients were then divided into 2 groups: those with (I-RHTN; n = 36) and those without (NI-RHTN; n = 93) myocardial ischemia. Echocardiography, 24-hour ambulatory BP monitoring (ABPM), and flow mediated dilation (FMD) were also evaluated. Thirty six (28%) patients had myocardial ischemia. There was no difference between groups regarding age, sex, biochemical parameters, office, and 24-hour ABPM levels. Patients in the I-RHTN group were more likely diabetic (31% vs. 11%; P < 0.05) and obese (75% vs. 40%; P < 0.001). Adjusting for age and body mass index, multiple logistic regression showed that diabetes (odds ratio (OR) = 6.5; 95% confidence interval (CI) = 1.06-40.14; P = 0.04), FMD (OR = 0.18; 95% CI = 0.07-0.41; P < 0.001), heart rate (OR = 1.23; 95% CI = 1.11-1.36; P < 0.001), left ventricular mass index (OR = 1.02; 95% CI = 1.01-1.04; P = 0.04), and microalbuminuria (OR = 1.02; 95% CI = 1.01-1.04; P = 0.002) were independent predictors of ischemia. In conclusion, there is a high prevalence of myocardial ischemia in patients with RHTN. Increased microalbuminuria, heart rate, endothelial dysfunction, and left ventricular mass can be useful to guide the investigation for myocardial ischemia in these high risk patients.

Characterization of Fiber Types in Different Muscles of the Hindlimb in Female Weanling and Adult Wistar Rats

We analyzed lesser diameter and distribution of fiber types in different skeletal muscles from female Wistar rats using a histoenzymology Myofibrillar Adenosine Tri-phosphatase (mATPase) method. Fragments from muscles were frozen and processed by mATPase in different pH. Adult and weanling rat soleus muscles presented a predominance of type I fibers and larger fiber diameters. In the plantar muscle in adult rats, the type IIB fibers demonstrated greater lesser diameter while in the weanling animals, types I and IIB fibers were larger. The plantar muscle of animals of both ages was composed predominantly of the type IID fibers. The type IID fibers were observed in similar amounts in the lateral gastrocnemius and the medial gastrocnemius muscles. Type IIB fibers showed predominance and presented higher size in comparison with other types in the EDL muscle. The present study shows that data on fiber type distribution and fiber lesser diameter obtained in adult animals cannot always be applied to weanling animals of the same species. Using the mATPase, despite the difficult handling, is an important tool to determine the different characteristics of the specific fibers in the skeletal muscle tissue.

Vascular relaxation of canine visceral arteries after ischemia by means of supraceliac aortic cross-clamping followed by reperfusion

Background: The supraceliac aortic cross-clamping can be an option to save patients with hipovolemic shock due to abdominal trauma. However, this maneuver is associated with ischemia/reperfusion (I/R) injury strongly related to oxidative stress and reduction of nitric oxide bioavailability. Moreover, several studies demonstrated impairment in relaxation after I/R, but the time course of I/R necessary to induce vascular dysfunction is still controversial. We investigated whether 60 minutes of ischemia followed by 30 minutes of reperfusion do not change the relaxation of visceral arteries nor the plasma and renal levels of malondialdehyde (MDA) and nitrite plus nitrate (NOx). Methods: Male mongrel dogs (n = 27) were randomly allocated in one of the three groups: sham (no clamping, n = 9), ischemia (supraceliac aortic cross-clamping for 60 minutes, n = 9), and I/R (60 minutes of ischemia followed by reperfusion for 30 minutes, n = 9). Relaxation of visceral arteries (celiac trunk, renal and superior mesenteric arteries) was studied in organ chambers. MDA and NOx concentrations were determined using a commercially available kit and an ozone-based chemiluminescence assay, respectively. Results: Both acetylcholine and calcium ionophore caused relaxation in endothelium-intact rings and no statistical differences were observed among the three groups. Sodium nitroprusside promoted relaxation in endothelium-denuded rings, and there were no inter-group statistical differences. Both plasma and renal concentrations of MDA and NOx showed no significant difference among the groups. Conclusion: Supraceliac aortic cross-clamping for 60 minutes alone and followed by 30 minutes of reperfusion did not impair relaxation of canine visceral arteries nor evoke biochemical alterations in plasma or renal tissue.