| IntroductionPeripheral arterial disease (PAD) is a major cause of acute and chronic vessel illness and is associated with disability and reduced quality of life. The management of patients with PAD consists of life-style modifications and pharmacotherapy addressing the risk factors to minimize the risk for disease progression and mortality in myocardial infarction and stroke. Symptomatic invasive treatment consists of surgical or endovascular revascularization. Unfortunately, about20-30%of patients with PAD cannot be treated by any of these methods and the only option for them is often amputation. Therefore, a novel therapeutic modalities are needed to treat these patients. Promotion of collateral artery growth (arteriogenesis) is a recently proposed concept, which has brought a new therapeutic strategy for PAD in the past few years.Arteriogenesis is defined as growth of preexisting arteriolar connections into true collateral arteries. It relies on a complex combination of increased shear stress, different growth factors, cytokines, proteolytic enzymes and initial local inflammation. FSS (fluid shear stress) is the initiating molding force in arteriogenesis, which leads to further development in the shaping of the peripheral collateral circulation. The formation of a collateral circulation after an arterial occlusion correlates well with the calculated increase in FSS because of the increased collateral flow caused by the pressure decrease along pre-existent collaterals. Pipp F et al. reported that high levels of FSS, caused by artificial arteriovenous shunt between the distal stump of the occluded femoral artery and its accompanying vein, can produce a strong arteriogenic response.Drag-reducing polymers (DRPs)are long-chain, blood soluble macromolecules that possess a molecule weight over106Da and a relatively linear structure. Numerous studies have documented that continuous intravenous administrastion of Polyethelen oxide (PEO, one of drag reducing polymers) can improve ischemic tissue perfusion, and several studies showed that DRPs (drag reducing polymers) inhibit the development of atherosclerosis by increasing shear stress in areas normally exposed to low shear stress. We also found there is an30%increasement of arterial wall shear stress during PEO infusion. Adding to our preliminary experimental data, we hypothesized that DRPs can also promote arteriogenesis by FSS increament. The present experiments would be therefore undertaken to test the hypothesis, and to investigate the underlying mechanism in an ischemic hindlimb models of chronic femoral occlusion.PartⅠ Effects of Drag-reducing polymers on hemodynamics of abdominal aorta and hindlimb vascular resistance of chronic ischemia hindlimb ratsObjectiveEvaluation of the effect with chronic administration PEO on abdominal aorta blood flow and vascular resistance in rats hind limb ischemia to determine the relationship between energy efficiency of drag reduction, for further methodological foundation of chronic administration model in the study.Methods1.Preparation of DRPsDRPs were prepared by using a commercial polyethylene oxide (PEO) with an average molecule weight (MW) of5×106Da. The polymer was dissolved in normal saline. The solution was then dialyzed against saline for24hours using a membrane with50kDa molecular weight (MW) cutoff to remove low MW impurities and stored at4℃.8h prior to the experiment, the PEO solution was diluted with normal saline to a final concentration of50μg/ml.2. Preparation of animal modelWistar rats were subjected to unilateral femoral artery ligation under anesthesia with intraperitoneal administration of pentobarbital sodium (45mg/kg). A vertical incision was made along the inner right hindlimb. The right femoral artery was exposed, ligated with5-0silk ligatures without damaging the vein and nerve. The left limb served as an internal control for each rat. Jugular veins of rats were separated and inserted catheter which was fixed with sutures in multiple subcutaneous and fixed the boot to the back incision. Caged rats were divided and jugular vein catheter wash piped daily with0.1ml of heparin saline (10iu/ml). The rats were administrated PEO or saline through the infusion channel at a constant rate of about1ml/h for30minutes according to the assigned treatment group every two day in a conscious unrestrained state.3. Hemodynamic MonitoringTwelve rats were anesthetized and randomly divided into two groups. An ultrasonic flow probe was placed around the abdominal aorta (5mm above the common iliac artery) to measure blood flow. Vascular resistance was calculated as ΔP/abdominal aortic flow, where ΔP=iliac artery pressure-iliac vein pressure.Results1. Effect of abdominal aortic blood flow:there is an interaction effect between two groups in abdominal aortic blood flow (F=3.076, P=0.000); statistically significant differences between groups (F=85.196, P=0.000), the effect of different treatment groups suggesting differences; each time point difference was statistically significant (F=3.47, P=0.000) indicate different between the two groups of blood flow at different times.Further comparison found:two groups of rats under basal blood flow in the abdominal aorta was no significant difference (4.47±0.60vs4.47±0.95ml/min, t=0.016, P=0.988). DRA group of abdominal aortic blood flow was significantly increased in the administration process, reached a peak of about8min, and maintain a platform, followed by a slow decline in blood flow changes each time point were statistically significant (F=5.995, P=0.000). Saline group during administration of abdominal aortic blood flow change was not statistically significant (F=0.158, P=1.000)2. Effect of the hind limb vascular resistance:two groups of rats hind limb vascular resistance does not exist interaction effect (F=1.117, P=0.359); between-group difference was statistically significant (F=22.17, P=0.000), the effect of different treatment groups suggesting differences; each time difference was not statistically significant (F=1.165, P=0.135) suggesting no hind limb vascular resistance at different times change significantlyFurther comparison, two groups of rats under basal hindlimb vascular resistance was no significant difference (0.44±0.10vs0.43±0.04mmHg·min/ml, t=0.29, P=0.78). PEO group of drag reduction effect is obvious, hindlimb vascular resistance decreased significantly during administration, about14-18min to achieve maximum effect and maintain a stable, slow recovery after the withdrawal, the resistance changes each time point was not statistically significant (F=1.632, P=0.173). Saline group hindlimb vascular resistance changed little in the administration process, no statistical significance (F=0.216, P=0.95).Between two groups, the hindlimb vascular resistance showed no significant difference at baseline (0.44±0.10vs0.43±0.04mmHg·min/ml, F=0.08, P=0.78). PEO group of drag reduction effect is obvious, hindlimb vascular resistance decreased significantly during administration, at about14-18min to achieve maximum effect and maintain a stable, then slow recovery after the withdrawal. The resistance changes each time point was not statistically significant (F=1.632, P=0.173). Saline group hindlimb vascular resistance changed little in the administration process with no statistical significance (F=0.216, P=0.95); There were significant differences between two groups in the trends of hind vascular resistance (F=22.27, P=0.000).ConclusionsPEO can increase abdominal aorta blood flow in rats chronic limb ischemia model and promote local blood flow, while reducing the vascular resistance.PartⅡ Effects of Drag-reducing polymers on Arteriogenesis in chronic ischemia hindlimb ratsObjectiveEvaluation effect of PEO on chronic ischemic skeletal muscle microcirculation with ultrasound contrast, and combined with pathology, immunohistochemistry and microCT technical. Evaluate arteriogenesis effect on chronic limb ischemia model from different perspectives.With saline control group, assessment of different formation between angiogenesis and arteriogenesis, and explore different promotion effect of PEO.Methods1. Preparation of DRPs and Preparation of animal model was same with Part Ⅰ.2. Contrast Echocardiography of hindlimb skeletal muscle microcirculation and data analysisUse Sequoia512echocardiography in the preoperative0day and postoperative1,7,14,21day separately to evaluate the microcirculation perfusion in ischemic hindlimb. Ultrasound probe fixed on the middle hindlimb at short axis with coherent pulse sequence (CPS) and time trigger imaging. Recording A, β and A×β value for offline use MCE software. Contrast agent according to a function of the intensity and time intervals of local tissue Y=A (1-e (-β*t)) of micro bubbles filled reperfusion curve. Contrast agent refill rate (β), reflects the flow rate of the local tissue in capillaries; skeletal platform acoustic intensity (A) reflecting the local microcirculation blood volume; Product A×β reflects local tissue blood flow.3. PathologyAt0day,7days,14days and21days before and after surgery, respectively animals were sacrificed with right hind limb adductor collected. HE staining, α-actin antibody and CD31antibody were used in Immunohistochemistry.4. Micro angiography21days after surgery, Rats were anesthetized and The infrarenal abdominal aorta was ligated proximally and cannulated distally with a PE-50catheter to the abdominal aorta1cm proximal to the iliac bifurcation. The lower hindlimbs were perfused with2mL of warm heparinized saline (10U/mL). Postmortem micro angiography was then performed. Nitroglycerine was delivered through, followed by a perfusion of2mL saline. After a period of1minute,3ml of60%iodinated contrast medium was manually injected. Images were acquired with an X-ray mammography system from the level of the left common iliac artery to the distal end of the ischemic limb to image the morphological development of collateral vessels.Results1. PEO group perfusion recovery was significantly faster than saline group. In the first14days, The blood flow (A×β) in PEO group has been significantly faster in the saline group (4.94±0.82vs3.46±1.13, t=2.596, P=0.027). For21days, ischemic hind limb blood flow PEO group is higher than the saline control group, the blood flow is restored to the PEO groups preoperatively (90.9±17.9)%, while the saline group restored only to (73.3±29.4), between two groups, there was a significant difference (5.57±1.15vs4.09±1.04dB/s, t=2.343, P=0.001); skeletal muscle blood volume (A) compared with the saline group at each time point showed no significant change ((F=0.045, P=0.836); blood flow velocity (P) presented similar trend with blood flow (A×(3), compared with saline group in the first14days and21days was significantly higher,(0.22±0.04vs0.16±0.04s-1, t=2.666, P=0.024) and (0.26±0.05vs0.19±0.05s-1, t=2.734, P=0.021) respectively, the difference was statistically significant.2. HE staining:Ischemic hindlimb skeletal muscle cells were normal,there was no congestion, edema in muscle cells. In both groups we can see inflammatory cell infiltration, and was much higher in PEO group than the saline group.3. α-actin immunohistochemistry:Compared with saline group, the number of small arteries show no significant difference in PEO group at day7(29.83±6.8vs27.42±5.72, t=0.666, P=0.52). In the14day and21day increased respectively (40.78±5.45vs33.01±4.66个mm2, t=2.653, P=0.024) and (47.10±6.04vs3.18±6.47个/mm2,t=2.588, P=0.027). 4. CD31capillary density:For7days, compared with preoperative day0, the number of capillaries positive staining in PEO group was significantly increased (359.17±21.74vs312.33±21.42, P=0.006).14days after surgery showed slow downward trend,there was no significant difference in the21days with preoperative dayO (314.17±26.19vs312.33±21.42, P=0.906). Comparison of various points of time no significant difference between the two groups (P>0.05).5. CT angiography showed after21days of treatment, PEO group collateral vessels vascular score was significantly higher than the saline group (1.42±0.16vs1.16±0.16, t=2.83, P=0.018), there was statistically significant.Conclusions1. PEO can promote angiogenesis and arterial formation process in chronic ischemic hind limbs, and may provide a new safe and effective means for the treatment of peripheral vascular disease.2. PEO can promote angiogenesis in ischemic limbs, which acts mainly through the artery promotion rather than to promote capillaries in the process.PartⅢ The possible mechanism of Drag-reducing polymers on Arteriogenesis in rats with chronic limb ischemiaObjectiveEvaluation Cofilinl and Cofilin2genes, monocyte adhesion factor (MCP-1) and the adhesion of monocytes with administration of PEO by RT-PCR and scanning electron microscopy in chronic hindlimb ischemia and to further explore the possible mechanisms of PEO promotes arteriogenesis.Methods1. Preparation of DRPs and Preparation of animal model was same with Part I.2. Determination of muscle Cofilin1mRNA and Cofilin2mRNA expressionAfter21days of treatment, the muscle samples were collected, Cofilin1mRNA, Cofilin2mRNA expression levels was using real-time PCR for relative quantitative detection.3. Determination of MCP-1mRNA expression was same with part of Cofilin1 mRNA and Cofilin2mRNA’s determination.4. SEM:After treatment of21days, artery tissues were examined with scanning election microscope.Results1. Cofilin1mRNA expression in PEO group compared with the saline group was significantly higher (1.81±0.69vs1.00±0.00, t=2.88, P=0.035).2.Compared with the saline group, Cofilin2mRNA expression was lower in PEO group (0.64±0.33vs1.00±0.00, t=2.62, P=0.026), there was a significant difference between the two groups.3. MCP-1mRNA significantly increased in PEO group than in saline group (2.01±0.57vs1.11±0.42, t=3.09, P=0.01).4. Scanning electron microscopy shows that on the intima surface of PEO group, we can see a lot of monocytes adhesion, gathered into a group and some of them invasion from intima into media. Saline group:there was fewer monocyte adhesion and cell debris on visible surface, and no accumulation of monocytes can be seen.ConclusionPEO can upregulate the expression of Cofilin1mRNA expression and downregulate Cofilin2mRNA expression in rats with chronic ischemic hindimb. It promote arteriogenesis possibly through participating in smooth muscle cell migration and proliferation, and therefore promote collateral vessel development and maturation process.PEO may act on endothelial cells by increasing the shear stress mediated by a mechanical force-biological effect, and then increase the expression of MCP-1, the ability of adhesion and invasion of monocytes, in order to promote the process of arteriogenesis. |
PDF Full Text Request