Therapeutic angiogenesis of bone marrow mononuclear cells (MNCs) and peripheral blood MNCs: transplantation for ischemic hindlimb

We investigated bone marrow mononuclear cells (BM-MNCs) and peripheral blood mononuclear cells (PB-MNCs) for therapeutic angiogenesis in the ischemic hindlimb. BM-MNCs were isolated and injected into ischemic skeletal muscles in mice. Laser Doppler and histological evaluation were performed after the surgical procedure. Fifteen patients suffering from critical lower limb ischemia received subcutaneous injections of recombinant human granulocyte colony-stimulating factor (G-CSF) to mobilize progenitor cells, and PB-MNCs were harvested and transplanted directly into the ischemic limb. Endothelial cells derived from BM-MNCs were plated, then induced to form three-dimensional networks by invading a Matrigel. Four weeks after implantation of BM-MNCs, laser Doppler analysis showed that the blood flow ratio was significantly increased (0.67 +/- 0.02 vs. 0.44 +/- 0.02). Alkaline phosphatase and immunohistochemical analyses showed that capillary density was significantly increased (95.25 +/- 0.07% vs. 39.6 +/- 0.04%). Two months after implantation of PB-MNCs, in both subgroups, ankle-brachial index values, walking distance, pain scale, and transcutaneous oxygen pressure (TcO(2)) were significantly improved (p < 0.005). A total of six of 15 limb ulcers of transplanted patients were healed after cell transplantation. BM-MNC implantation was able to induce functional angiogenesis in mice ischemic hindlimb. This clinical trial shows that G-CSF-based PB-MNC transplantation is a feasible treatment for the ischemic hindlimb.     

The Prostacyclin Analog Beraprost Sodium Augments the Efficacy of Therapeutic Angiogenesis Induced by Autologous Bone Marrow Cells

Implantation of autologous bone marrow (BM) mononuclear cells (MNCs) has been shown to augment neovascular formation in ischemic tissues in experimental animals and in humans. Prostaglandin derivatives improve the symptoms of patients with critical limb ischemia, possibly by their vasodilating and antiplatelet actions. We therefore examined whether therapeutic angiogenesis by implantation of autologous BM-MNCs would be enhanced by beraprost sodium (BPS), using a rabbit ischemic hindlimb model. Ischemia was induced by surgical resection of the left femoral artery. Twenty-five New Zealand white rabbits were divided into four groups. The first group (BM group, n = 4) received autologous BM-MNCs (2 × 10⁶/animal) implanted into the ischemic tissue 1 week after limb ischemia. The second group (BM+BPS group, n = 8) received BPS injected into the dorsal skin (300 μg/kg daily) starting 1 week before limb surgery. This group received BM-MNC implantation 1 week after surgery. Daily injection of BPS was continued until the end of the protocol. The third group (BPS group, n = 8) received BPS injected into the dorsal skin (600 μg/kg daily) starting 1 week before limb surgery. The fourth group received saline as a control (n = 4). The extent of angiogenesis in the ischemic hindlimb was assessed using the angiographic score (AS), ischemic/normal limb calf blood pressure ratio (CBPR), and tissue capillary density. Four weeks after limb ischemia, the ischemic/normal CBPR was highest in the BM+BPS group, followed by the BPS, BM, and control groups (0.56 ± 0.16, 0.51 ± 0.25, 0.44 ± 0.15, and 0.30 ± 0.10, respectively). The AS was also the greatest in the BM+BP group, followed by the BM, BP, and S group (1.63 ±0.21, 1.31 ± 0.25, 1.26 ± 0.21 and 0.80 ± 0.10, respectively). The TCD was greatest in the BM+BP group, followed in by the BM, BP, and S group? (46 ± 4.1, 34 ± 0.7, 33 ± 6.9, and 19 ± 1.8 per field, respectively). BP treatment is an effective means to enhance the efficacy of therapeutic angiogenesis induced by autologous BM-MNCs implantation in ischemic hindlimb tissues.     

Functional outcome of new blood vessel growth into ischemic skeletal muscle

PURPOSE: The administration of angiogenic growth factors and the transfer of well-vascularized tissues have been shown to induce development of new blood vessels in ischemic muscle. The functional significance of these new vessels is unknown. The hypothesis of this study is that the transfer of vascularized muscle and the local infusion of basic fibroblast growth factor (bFGF) synergistically improve contractile function of ischemic skeletal muscle. METHODS: Twenty-six rabbits were divided into four groups. An ischemic hindlimb was created in each by ligating the right common iliac artery. The flap + bFGF group (n = 6) had transposition of a contralateral rectus muscle flap onto the thigh. Additionally, bFGF (3 ng/h) was continuously infused at the flap-thigh interface. In the flap group (n = 6), a similar muscle flap was created, but carrier solution was infused at the interface. In the bFGF group (n = 6), no muscle flap was created; instead, bFGF (3 ng/h) was infused into the external iliac artery of the ischemic limb. In the control group (n = 8), carrier solution was infused into the external iliac artery (no flap, no bFGF). After 1 week, the soleus muscle was isolated and stimulated. Maximum twitch tension, the fatigue index (force of contraction after 2 minutes of continuous stimulation/initial force of contraction), maximum recovery, and the number of limbs recovered (ie, limbs that achieve a force of contraction during the recovery period of > 75% of the force of the initial contraction at the start of continuous stimulation) were recorded. Blood vessel density (number of vessels per ***) was determined by immunostaining the soleus muscle with anti-alpha-actin antibody. RESULTS: All values were indexed to the contralateral normal limb. The flap + bFGF group showed significant improvement versus the control group in maximum twitch tension (1.07 +/- 0.13 vs 0.63 +/- 0.12, P < .05), maximum recovery (0.94 +/- 0.05 vs 0.58 +/- 0.05, P < .05), and the number of limbs recovered (5/5 vs 0/6, P < .05). This improved function correlated with increased vessel density (flap + bFGF group, 1.44 +/- 0.11 vs control group, 0.72 +/- 0.01, P < .05). CONCLUSION: Reperfusion of an ischemic limb with a well-vascularized muscle flap and local bFGF infusion promoted increased blood vessel density in distal ischemic muscle. This increased vascularity was associated with restoration of otherwise impaired muscle function. Improved function occurred rapidly (1 week). A transposed muscle flap provided a functional blood supply to the site of maximum ischemia; this could be used to salvage otherwise nonreconstructible ischemic limbs.     

Therapeutic angiogenesis induced by local autologous bone marrow cell implantation

BACKGROUND: Therapeutic angiogenesis was induced by local autologous bone marrow cell implantation (BMCI) in ischemic hindlimb or ischemic heart models in rats. This study was designed to investigate the toxicity and therapeutic potency of local BMCI using a chronic coronary occlusion model in dogs. METHODS: The canine chronic coronary occlusion model was created by ligating of the left anterior descending artery (LAD). The myocardium in the left ventricle was divided into distinct normal, marginal, and infarction areas 30 days after LAD ligation. Each area was injected at two locations, with either 2 x 10(7) bone marrow cells (n = 7, BMCI group) or 0.1 mL phosphate-buffered saline (PBS) only (n = 7, PBS group), respectively. Hemodynamics were evaluated by a single ultrasonic transducer and echocardiography before and 30 days after the treatment. Angiogenesis was evaluated by vessel count 30 days after the treatment. The toxicity of BMCI treatment was also evaluated in 8 normal dogs by following changes in electrocardiography (ECG), echocardiography, local histology, and systemic biochemistry indexes. RESULTS: There was a significantly higher percentage of wall thickening in the marginal area in the BMCI group than in the PBS group 30 days after treatment (14.5 +/- 2.28 versus 8.1 +/- 3.00, p = 0.002). Significantly more microvessels were observed in the marginal area in the BMCI group than in the PBS group 30 days after treatment (127.7 +/- 20.1 versus 88.0 +/- 10.2/field, p = 0.0007). No systemic or local toxicity was found following BMCI treatment in the acute or chronic phases. CONCLUSIONS: BMCI treatment improved local wall thickening dynamics, presumably due to the angiogenesis induced by the treatment. This indicates that it might be a safe and effective therapy for ischemic heart disease.     

Placental growth factor provides a novel local angiogenic therapy for ischemic cardiomyopathy

BACKGROUND: Heart failure occurs predominantly due to coronary artery disease and may be amenable to novel revascularization therapies. This study evaluated the effects of placental growth factor (PlGF), a potent angiogenic agent, in a rat model of ischemic cardiomyopathy. METHODS: Wistar rats underwent high proximal ligation of the left anterior descending coronary artery and direct injection of PlGF (n = 10) or saline as a control (n = 10) into the myocardium bordering the ischemic area. After 2 weeks, the following parameters were evaluated: ventricular function with an aortic flow probe and a pressure/volume conductance catheter, left ventricular (LV) geometry by histology, and angiogenesis by immunofluorescence. RESULTS: PlGF animals had increased angiogenesis compared to controls (22.8 +/- 3.5 vs. 12.4 +/- 3.2 endothelial cells/high-powered field, p < 0.03). PlGF animals had less ventricular cavity dilation (LV diameter 8.4 +/- 0.2 vs. 9.2 +/- 0.2 mm, p < 0.03) and increased border zone wall thickness (1.85 +/- 0.1 vs. 1.38 +/- 0.2 mm, p < 0.03). PlGF animals had improved cardiac function as measured by maximum LV pressure (95.7 +/- 4 vs. 73.7 +/- 2 mmHg, p = 0.001), maximum dP/dt (4206 +/- 362 vs. 2978 +/- 236 mmHg/sec, p = 0.007), and ejection fraction (25.7 +/- 2 vs. 18.6 +/- 1%, p = 0.02). CONCLUSIONS: Intramyocardial delivery of PlGF following a large myocardial infarction enhanced border zone angiogenesis, attenuated adverse ventricular remodeling, and preserved cardiac function. This therapy may be useful as an adjunct or alternative to standard revascularization techniques in patients with ischemic heart failure.     

Angiogenic response induced by mechanical transmyocardial revascularization.

BACKGROUND: Angiogenesis is the proposed mechanism of transmyocardial revascularization. We evaluated mechanical transmyocardial revascularization in a chronically ischemic porcine model by measuring myocardial angiogenic response. METHODS: Ameroid constrictors were implanted 6 weeks before mechanical transmyocardial revascularization. Group I (n = 5) and group II (n = 3) animals received 30 punctures with an 18-gauge needle and samples were harvested at 1 and 4 weeks, respectively, after the operation. Group III (n = 5) had sternotomy only and served as the control group. Myocardial samples were immunohistochemically stained for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF-beta) using specific antibodies. Growth factor expression was quantified by means of computer-assisted morphometry. Vascular density was assessed by immunohistochemical stain for VEGF and factor VIII. RESULTS: Compared with group III, increased angiogenic factor levels were found in group I (VEGF 0.47 +/- 0.03 mm(2) vs 0.05 +/- 0.05 mm(2), P =.000; bFGF 0.67 +/- 0.14 mm(2) vs 0.03 +/- 0.03 mm(2), P =. 000; TGF-beta 1.40 +/- 0.18 mm(2) vs 0.09 +/- 0.06 mm(2), P = 0.000), and in group II (VEGF 0.34 +/- 0.06 mm(2) vs 0.05 +/- 0.05 mm(2), P =.003; bFGF 0.06 +/- 0.02 mm(2) vs 0.03 +/- 0.03 mm(2), P =.135; TGF-beta 0.28 +/- 0.09 mm(2) vs 0.09 +/- 0.06 mm(2), P =.042). Vascular densities after mechanical transmyocardial revascularization were also increased (group I, VEGF stain 8.1 +/- 0. 6 vs 1.1 +/- 0.5, P =.000; factor VIII stain 5.1 +/- 2.7 vs 0.4 +/- 0.3, P =.018; group II, VEGF stain 1.9 +/- 0.5 vs 1.1 +/- 0.5, P = 0. 107; factor VIII stain 2.3 +/- 0.4 vs 0.4 +/- 0.3, P =.004). CONCLUSIONS: Mechanical transmyocardial revascularization is associated with increased angiogenic factor expression and concomitant neovascularization at up to 4 weeks. These changes are indistinguishable from those of laser transmyocardial revascularization. Myocardial perfusion studies are needed to establish the functional significance of these angiogenic changes.     

Successful orthotopic pig heart transplantation from non-heart-beating donors.

BACKGROUND: With the aim to expand the severely limited donor pool by use of non-heart-beating donors we developed a technique for successful transplantation of hearts after 30 minutes of normothermic ischemia without donor pretreatment. METHODS: In control groups hearts were transplanted in a conventional fashion using crystalloid cardioplegia (Group I, n = 6) or BCP (Group II, n = 8) for induction of cardiac arrest. In the ischemic groups hearts were harvested after 30 minutes of normothermic ischemia, perfused with blood cardioplegia (BCP) (Group III, n = 9) or BCP containing the Na(+)-H(+)-exchange inhibitor HOE 642 (Group IV, n = 8) and transplanted orthotopically. RESULTS: All animals could be weaned from cardiopulmonary bypass. Low dose inotropic support was necessary in the ischemic groups only. Recovery of the maximal left ventricular stroke work index (LVSWImax) in Groups I vs II was 62.6+/-19.6% vs 73.3+/-23.3% (NS), maximal right ventricular stroke work index (RVSWImax) averaged 61.1+/-18.8 vs 87.8+/-31.7% (NS) as compared to the preoperative level. In the ischemic groups (III vs IV) LVSWImax was 27.3+/-11.7 vs 59.5+/-32.4% (p = 0.038), RVSWImax was 27.4+/-20.9 vs 64.2+/-46.6% (NS). CONCLUSIONS: The results indicate that (a) successful pig heart transplantation after 30 minutes of normothermic ischemia is possible without donor pretreatment, and (b) that HOE 642 improves posttransplant LVSWImax significantly.     

Coronary atherosclerosis in cardiac transplant patients treated with total lymphoid irradiation.

BACKGROUND: Multiple episodes of rejection following cardiac transplantation have been associated with an increased incidence of coronary atherosclerosis. Total lymphoid irradiation (TLI) has been shown to be a successful treatment for persistent allograft rejection, but its effect on coronary arterial disease has yet to be evaluated. METHODS: From 1987 to 1999, 40 patients required TLI for persistent or recurrent allograft rejection following heart transplantation. Each patient's (Group 1, n = 31) post-transplant coronary angiograms were examined and compared with those of a control group (Group 2, (n = 32) matched for time of transplantation. Degree of coronary stenosis was assessed on a 6-point scale. All patients received induction therapy (rabbit anti-thymocyte globulin or OKT3) and standard triple immunosuppressive therapy. TLI (80 cGy x 10 fractions) was used for the treatment of recurrent or persistent rejection on the basis of clinical indications. Actuarial survival, number and treatment of rejection episodes, and severity of coronary artery disease were compared in each group. RESULTS: Recipient gender, age, race and cytomegalovirus (CMV) status at time of transplant, along with donor gender, CMV status and graft ischemia time, were similar in both groups. Group 1 donor age was younger than that of Group 2 (22.2 +/- 11.2 vs 31.5 +/- 13.6 years, p = 0.004), and the indication for surgery in Group 1 patients was more likely to be ischemic heart disease (15 of 31 vs 6 of 32, p = 0.02). Mean follow-up was 5.7 +/- 3.5 years in Group 1 vs 6.9 +/- 3.8 in Group 2 (p = NS). Group 1 had more rejection episodes (4.4 +/- 2.2 vs 2.3 +/- 2.0, p = 0.0002) and more steroid treatments (9.78 +/- 4.0 g vs 5.14 +/- 4.7 g, p < 0.0001), but less coronary artery disease compared with Group 2 (p = 0.035). CONCLUSIONS: Despite multiple episodes of rejection, patients treated with TLI after cardiac transplant appear to develop less coronary atherosclerosis than appropriately matched controls.     

Therapeutic angiogenesis using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model

BACKGROUND: We evaluated the effect of autologous marrow stromal cells (MSCs) on neovascularization and blood flow in an animal model of chronic limb ischemia. METHODS: Chronic hind limb ischemia was created by ligating the left common iliac artery of male Lewis rats. Three weeks after ligation, 5.0 million LacZ+MSCs (n = 10) or culture medium (n = 10) were injected into the anteromedial muscle compartment of the left thigh. At 4 and 6 weeks after injection, half the animals (n = 5) from each group underwent femoral artery ultrasonic blood flow measurements of the ischemic and nonischemic limbs to obtain a flow ratio. The animals also underwent angiography and measurements of blood vessel density and arteriolar density. Qualitative histologic assessment of the limb muscles was performed. RESULTS: LacZ+MSCs were found to differentiate into endothelium (F VIII+), vascular smooth muscle (positive a-smooth muscle actin), skeletal muscle (positive desmin), and adipocytes. Ischemic hind limbs where MSCs were implanted had greater vascular density and arteriolar density than control limbs (p < 0.001). Femoral artery flow index (left femoral artery flow/right femoral artery flow) was 0.89 +/- 0.12 and 0.90 +/- 0.06 for rats injected with MSCs measured at 4- and 6-weeks, respectively, compared with 0.50 +/- 0.15 and 0.50 +/- 0.10 for the control rats (p < 0.001). Angiography demonstrated reconstitution of the left femoral artery in rats that received MSC implantation through pelvic and abdominal wall collateral formation. CONCLUSIONS: Local MSC implantation induces a neovascular response resulting in a significant increase in blood flow to the ischemic limb. Marrow stromal cells are also capable of spontaneously regenerating the various components of muscular tissues.     

Comparison of vascular endothelial growth factor and fibroblast growth factor-2 in a swine model of endothelial dysfunction.

OBJECTIVE: Growth factor based angiogenesis, with or without cell therapy, is a promising therapeutic modality for patients with coronary artery disease. We compared the relative efficacies of surgically delivered vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in a swine model of hypercholesterolemia-induced endothelial dysfunction which captures many of the pathophysiologic abnormalities of human coronary disease. METHODS: Yucatan mini-swine (20-30 kg), fed a high cholesterol diet (total 20 weeks), underwent circumflex ameroid placement to create chronic myocardial ischemia, followed three weeks later by perivascular administration of VEGF (2 microg; n=6), FGF-2 (100 microg; n=6), or placebo (n=7) in the ischemic territory. Normocholesterolemic animals (n=7) served as controls. Four weeks later, endothelial function, collateral-dependent perfusion, as well as myocardial protein and mRNA levels of angiogenic mediators were assessed. RESULTS: Endothelial dysfunction was observed in all hypercholesterolemic animals as impaired microvessel relaxation in response to adenosine diphosphate and VEGF. VEGF administration improved baseline-adjusted collateral-dependent perfusion at rest (-0.03+/-0.05 vs -0.12+/-0.04, VEGF vs placebo, p=0.09), but FGF-2 delivery caused a significantly greater improvement in perfusion compared to either group (+0.15+/-0.03, p<0.05 vs HC-placebo and HC-VEGF) at rest. Molecular analysis revealed increased eNOS expression (135%+/-8%, p=0.03 vs placebo) in all growth factor treated animals and increased expression of FGF-2 receptor, FGFR1 (65+/-26%, p=0.04 vs placebo), in FGF-2 treated animals. No significant changes were demonstrated in other angiogenic mediators including Akt, Syndecan-4. CONCLUSIONS: In the setting of hypercholesterolemic endothelial dysfunction, FGF-2 is more effective than VEGF at enhancing collateral-dependent perfusion and thus, may be a better candidate than VEGF for angiogenic therapy in patients with end-stage CAD.     

Frizzled A, a novel angiogenic factor: promises for cardiac repair.

OBJECTIVE: Frizzled A is a very recent protein expressed in the cardiovascular hood by cardiomyocytes and by endothelial cells. This protein plays key roles in vitro in vascular cell proliferation and is able to induce an in vivo angiogenic response. Regarding these properties, we assess the hypothesis that Frizzled A could act in the healing process after myocardial infarction. METHODS: To investigate the role of Frizzled A, we established a transgenic mouse line overexpressing the protein and developed a model of myocardial infarction by coronary artery ligation. RESULTS: The incidence of cardiac rupture after myocardial infarction was reduced in transgenic mice (6.5 versus 26.4% in controls, n=165; P<0.01). Infarct sizes were smaller in transgenic mice (18% of left ventricle circumference versus 28.1% in control at day 30; P<0.001; n=6) and the cardiac function was improved (3800 +/- 370 versus 2800 +/- 840 mmHg/s dp/dtmax in controls, -2800 +/- 440 versus -1800 +/- 211 dp/dtmin in controls at day 15; P<0.001; n=6). Early leukocyte infiltration had decreased in transgenic mice during the first week (103 +/- 59 versus 730 +/- 463 cells/mm2 in controls at day 7; P<0.001; n=6) and the apoptotic index was decreased by 50% at day 7. Capillary density in the scar was higher in transgenic mice (290 +/- 103 versus 104 +/- 43 vessels/mm2 in control at day 15; P<0.001) and vessels were more muscularized and mean lumen area was 3-fold higher (952 +/- 902 versus 313 +/- 350 microm2 in control; P<0.001). CONCLUSION: Overexpression of Frizzled A reduced the infarct size, improved cardiac recovery, modified inflammatory response and amplified angiogenesis. For these reasons, this protein would be of interest for cardiac surgeons using angiogenic therapy (as gene or protein injection) in ischemic heart diseases in non-revascularizable patients.     

Exercise-induced hyperemia unmasks regional blood flow deficit in experimental hindlimb ischemia

Many experimental models of hindlimb ischemia are characterized by spontaneous and rapid normalization of resting muscle blood flow (BF) rates which complicates the long-term evaluation of angiogenic therapies to reverse limb ischemia. We tested the hypothesis that peroneal nerve stimulation in an ischemic hindlimb would increase the oxygen (O(2)) demand and BF rate, thereby unmasking a severe blood flow deficit that is not apparent at rest. METHODS: Ischemia was induced in adult rats by ligation of the left common iliac, femoral arteries, and their branches. Peroneal nerves were stimulated to allow measurement of exercise-induced regional BF rates with fluorescent microspheres. Hemodynamics were monitored. Fluorescent microspheres were injected before and after 5 min of nerve stimulation 3, 10, and 24 days postischemia. The tibialis anterior (TA) and gastrocnemius (GC) muscles and skin were harvested and weighed, and fluorescence was measured. BF rate was calculated as milliters per minute per gram of tissue and compared to normal muscle and skin of unoperated control rats. In order to determine the accuracy of BF rate measurements in ischemic muscle when <400 microspheres was delivered per specimen, 3 rats were studied by simultaneous injection of 4 x 10(5) blue and 1 x 10(5) yellow-green fluorescent microspheres. The correlation coefficient between the number of different colored microspheres delivered was measured. RESULTS: The ischemia caused atrophy of the TA and GC muscles. The mean muscle mass of the ischemic TA and GC as a percentage of total body weight decreased over time vs control [TA 0.13 +/- 0.05% vs 0.25 +/- 0.03%, P < 0.05; GC 0.51 +/- 0.27% vs 0.70 +/- 0.07%, P = 0.07 at 24 days (24D)]. Despite clinical evidence of severe hindlimb ischemia in experimental groups, i.e., pressure sores, muscle atrophy, and weakness, resting BF rates were not significantly different from those of control. The BF rate of the TA was of 0.11 ml/min/g after 3D of ischemia, 0.14 ml/min/g after 10D, and 0.13 ml/min/g after 24D. The mean BF rate in normal muscle of unoperated controls was 0.16 ml/min/g (P > 0.05). However, the exercise-induced hyperemia in the skeletal muscle was significantly blunted in all of the ischemic groups. The unoperated control TA had a greater than 10-fold increase in BF to 1.95 ml/min/g in response to exercise while the ischemic TA had no increase in BF at 3D, 2-fold increase at 10D, and a 5-fold increase at 24D. Parallel findings were noted in the GC muscles. There was no significant difference in the BF rate in the skin. The accuracy of this microsphere technique in measuring very low BF rates found in ischemic muscle was supported by the significant correlation coefficient (r = 0.99) comparing two quantities of microspheres injected simultaneously. CONCLUSION: Despite clinical signs of severe hindlimb ischemia, resting BF rates in the ischemic groups were not significantly decreased. Peroneal nerve stimulation resulted in up to 10-fold increase in BF rate and unmasked a severe deficit in vascular reserve in the ischemic groups. Resting BF rate is not always an accurate reflection of the flow deficit in models of critical limb ischemia, and this model of exercise-induced hindlimb hyperemia may allow better long-term evaluation of angiogenic therapies designed to reverse critical limb ischemia.     

Serum enzyme levels during intestinal ischemia.

Because the intestinal mucosa is most sensitive to ischemia, serum levels of mucosal enzymes, such as diamine oxidase, may be most likely to indicate intestinal ischemia. Our aim was to compare serum levels of mucosal (diamine oxidase, alkaline phosphatase) and seromuscular (creatinine phosphokinase, lactic dehydrogenase, serum glutamic oxaloacetic transminase) enzymes during intestinal ischemia of varying extent and duration in dogs. Group 1 (n = 6) underwent sham laparotomy. Group 2 (n = 8) had 50% of the small intestine devascularized. Group 3 (n = 8) had the superior mesenteric artery occluded for 2 hours and released. Group 4 (n = 8) had the superior mesenteric artery ligated. Serum samples were obtained before and 2, 4, 8, and 24 hours after operation, and histologic specimens were examined at 4 hours. Creatinine phosphokinase levels became elevated within 4 hours of ischemic injury in group 2 (223 +/- 197 vs. 68 +/- 26, p less than 0.05) and group 4 (212 +/- 136 vs. 76 +/- 29, p less than 0.05). Significant elevation of serum enzymes levels, except diamine oxidase, occurred in groups 2, 3, and 4 at 24 hours, including those with normal histology after temporary superior mesenteric artery occlusion. Thus seromuscular enzymes, particularly creatinine phosphokinase, were more likely to be elevated during intestinal ischemia. Enzyme levels were not influenced by the extent and reversibility of the ischemic injury.     

Pulmonary complications of a roller pump right ventricular assist device

The pulmonary effects of a right ventricular assist device (RVAD) were evaluated in a model of ischemic right ventricular (RV) failure. The right coronary artery (RCA) was ligated for 240 min in 12 mongrel dogs. Group 1 (n = 5) was supported medically (iv fluids, epinephrine); Group 2 (n = 7) had an RVAD instituted 30 mins after RCA ligation, but no inotropic support was given. The RVAD was a standard roller pump providing right atrial to pulmonary artery flow which unloaded the RV. The ratio of area of infarction (AI) to area at risk (AR) of the RV was determined by vital dye staining. Total lung water (TLW) was determined by gravimetric analysis and expressed as milliliters per kilogram body weight. Throughout the experiment animals in Group 1 had significantly higher RV systolic pressures. Pulmonary vascular resistance was increased significantly in Group 2 at 4 hr (318% of baseline vs 33%). Mean pulmonary artery pressure increased significantly in Group 2 from 9.4 +/- 0.9 mm Hg at baseline to 21.0 +/- 5.0 mm Hg at 4 hr. Group 2 animals had a decreased AI/AR ratio (19 +/- 3 vs 57 +/- 9) and increased TLW (20 +/- 3 vs 9 +/- 1). Lung biopsies in Group 2 revealed perivascular, peribronchial, and intraalveolar hemorrhages that were not present in Group 1. In conclusion, a roller pump RVAD limits RV infarction but produces pulmonary hypertension, increases pulmonary vascular resistance, and creates pulmonary edema and hemorrhage in the process.     

Experimental hindlimb ischemia increases neutrophil-mediated matrix metalloproteinase activity: a potential mechanism for lung injury after limb ischemia

BACKGROUND: Acute limb ischemia initiates a systemic inflammatory response, including pulmonary polymorphonuclear leukocyte (PMN) sequestration and acute lung injury. Lung injury is partly attributed to release by PMN's of extracellular matrix (ECM) modifying metalloproteinases (MMPs). We hypothesized that acute hindlimb ischemia (HI) would increase MMP activity in the lung and other organs and that systemic neutrophil depletion before HI would block this effect. STUDY DESIGN: Seventeen FVB/N Tie2/LacZ-182 SATO female mice were randomly divided into four groups: HI + PBS (Group 1), HI + antineutrophil antibody (Group 2), HI + isotype matched control antibody (Group 3), and no HI + PBS (Group 4). HI was achieved by unilateral femoral artery ligation. Neutrophil depletion was confirmed. Three days postligation, lung, liver, and kidney were harvested. MMP-2 and -9 expression and activation (gelatin zymography) and membrane type-1 MMP (MT1-MMP, western blotting) were quantified by densitometry and NIH Image Analysis software. Statistical significance was determined with an analysis of variance. RESULTS: Zymograms revealed a 46% increase in pulmonary proMMP-9 in Group 1 versus Group 4 (6,107 +/- 472 [mean +/- SEM] densitometry units [DU] versus 3,287 +/- 675 DU, p < 0.05). A similar trend was observed for active MMP-9 (3,189 +/- 541 DU versus 1,417 +/- 927 DU, P = 0.16). Neutrophil depletion (Group 2) decreased proMMP-9 levels by 51% (2,996 +/- 314 DU versus 6,107 +/- 472 DU, p < 0.05) and active MMP-9 by 75% (810 +/- 444 DU versus 3,189 +/- 541 DU, p < 0.05) compared with Group 1. Active MMP-2 increased 51% after HI (Group 1, 3,230 +/- 86 DU versus Group 4, 1,599 +/- 327 DU, p < 0.05). Neutrophil depletion decreased the HI-induced activation of MMP-2 by 43% (Group 2, 1,829 +/- 471 DU versus Group 1, 3,230 +/- 86 DU, p < 0.05). CONCLUSIONS: HI increases pulmonary proMMP-9, active MMP-9, and active MMP-2 levels. Neutrophil depletion blocks this effect. These data suggest that acute limb ischemia leads to PMN-mediated changes in MMP activity.     

Comparison of intravenous ganciclovir and cytomegalovirus hyperimmune globulin pre-emptive treatment in cytomegalovirus-positive heart transplant recipients.

BACKGROUND: We compared the use of intravenous ganciclovir and cytomegalovirus hyperimmune globulin (CMVIG) as a pre-emptive treatment for cytomegalovirus (CMV)-positive heart transplant recipients. METHODS: Of 59 CMV-seropositive adult heart transplant recipients enrolled in Group 1, 37 tested positive for pp65 antigen within 12 weeks post-transplantation. These patients were randomized to receive either intravenous ganciclovir (n = 23) or CMVIG (n = 14). Group 2 included 133 CMV-seropositive heart transplant recipients who were not tested for CMV antigenemia and who received no anti-CMV therapy. RESULTS: CMV disease developed in 0 of 59 patients from Group 1, and in 27 of 133 patients (20%) in Group 2 (p = 0.0001). The incidence of superinfections was lower in Group 1 (0.28 +/- 0.46) than in Group 2 (1.10 +/- 1.33) (p = 0.01). The 2 groups did not differ with regard to incidence of rejection (0.7 +/- 0.9 in Group 1 vs 1.0 +/- 1.2 in Group 2; p = NS), transplant coronary artery disease at 1 year (14% in Group 1 vs 16% in Group 2; p = NS) or post-transplant lymphoproliferative disease (0% in Group 1 vs 2% in Group 2; p = NS). Ganciclovir and CMVIG therapies were associated with similar rates of rejection (0.52 +/- 0.6 with ganciclovir vs 0.50 +/- 0.60 with CMVIG; p = NS), superinfection (0.30 +/- 0.48 with ganciclovir vs 0.25 +/- 0.46 with CMVIG; p = NS), and transplant coronary artery disease at 1 year (13% with ganciclovir vs 14% with CMVIG, p = NS). CONCLUSIONS: The pre-emptive anti-CMV approach is superior to prophylaxis in CMV-seropositive heart transplant recipients. Both ganciclovir and CMVIG are equally effective.     

The effect of hyperbaric oxygen on reperfusion of ischemic axial skin flaps: a laser Doppler analysis.

This study evaluates the microvascular reperfusion of ischemic skin flaps with and without acute hyperbaric oxygen (HBO) treatment. Thirty-two axial pattern epigastric skin flaps (3 x 6 cm) in male Wistar rats were subjected to 8 hours of global ischemia by pedicle clamp occlusion. The rats were divided into the following control and two experimental groups: Control (n = 12) with ischemia, no HBO; Group 1 (n = 11) with HBO treatment (three 1.75-hour dives, 2.5 absolute atm, 100% O2) during ischemia; and Group 2 (n = 9) with HBO treatment (two 1.75-hour dives) immediately after ischemia. Laser Doppler flows were recorded in two distal standardized flap locations at 0.5, 2, 4, and 18 hours after reperfusion in control rats and Group 1 rats and at 18 hours only in Group 2 rats, using a Med-Pacific 6000 laser Doppler unit. Mean distal flap laser Doppler flows (mV) were Control: 0.5 hours = 23.2 +/- 11.9, 2 hours = 52.8 +/- 27.3, 4 hours = 53.6 +/- 32.1, 18 hours = 40.2 +/- 36.2; Group 1: 0.5 hours = 71.8 +/- 30.9 (p less than 0.05 vs. control), 2 hours = 74.3 +/- 27.3, 4 hours = 67.4 +/- 20.6, 18 hours = 79.1 +/- 40.3 (p less than 0.05 vs. control); and Group 2: 18 hours = 90.3 +/- 47.9 (p less than 0.05 vs. control). It is concluded that acute HBO treatment of ischemic rat skin flaps improves distal microvascular perfusion as measured by laser Doppler flowmetry. This effect is observed for HBO treatment given either during or immediately after prolonged global ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex-based differences in vascular repair with bone marrow cell therapy: relevance of regulatory and Th2-type cytokines

OBJECTIVES: There are differences in symptoms, risk stratification, and efficacy of pharmacological treatments between men and women with coronary artery disease (CAD). The results of clinical studies of cell therapy in CAD patients are mixed. The relevance of sex to response to cell therapy is unknown. We investigated sex-based differences in response to bone marrow mononuclear cells (BM-MNCs) in atherosclerotic apoliproprotein E-knockout (ApoE -/-) mice. METHODS: Twenty-three male and 27 female ApoE -/- mice fed on a high-fat diet received four intravenous BM-MNC injections (C57BL6/J mice) starting at 14 weeks of age; male or female BM-MNCs were administered. Thirteen male and 20 female atherosclerotic ApoE -/- mice received vehicle. Aortic plaque burden (%), recipient bone marrow progenitor cell profiles (FACS-LSR II, FlowJo) and 22 circulating cytokine panel (LINCOplex) were quantified and analyzed statistically (SSPS, P < or 5). RESULTS: Quantitative and semiquantitative results are presented. Increased G-CSF levels correlated with plaque reduction (r = -.86, P = .0004). G-CSF was clustered with IL-15. CONCLUSIONS: Female but not male BM-MNCs exhibited atheroprotection in male atherosclerotic ApoE -/- mice. Plaque lesions did not attenuate atherosclerosis in female ApoE -/- mice with BM-MNCs of either donor sex. An increase in regulatory and in Th2-type response may be required for atheroprotection. Sex-based differences in vascular repair have implications for cell therapy trials in CAD.     

Endogenous myocardial angiogenesis and revascularization using a gastric submucosal patch

BACKGROUND: The gastrointestinal submucosa physiologically produces angiogenic proteins. We examined whether these properties could lead to endogenous myocardial angiogenesis in a swine model of chronic ischemia. METHODS: Fifteen Yorkshire swine underwent ameroid constrictor placement around the circumflex artery and either lateral epicardial abrasion, creation of a gastroepiploic artery (GEA) based gastric patch, mucosal avulsion, transdiaphragmatic transfer, and apposition of the patch against the circumflex myocardial territory (number = 8; test animals), or lateral epicardial abrasion alone (number = 7; controls). Seven weeks later, lateral myocardial perfusion, endothelial cell density, and expression of VEGFR-1 and VE-cadherin were determined using isotope-labeled microsphere assays, immunohistochemistry, and immunoblotting, respectively. RESULTS: Microsphere assays showed equivalent lateral/anterior myocardial perfusion indices at rest (1.10 +/- 0.49 vs 0.95 +/- 0.23, test vs control animals; p = 0.54), but higher perfusion in test animals versus controls during pacing (1.05 +/- 0.29 vs 0.69 +/- 0.09, test vs controls; p = 0.02). Increased myocardial endothelial cell density (42.6 +/- 8.5 vs 26.1 +/- 11.6 cells per 3850 microm2, test vs controls; p = 0.02) and expression of VE-cadherin (3.10 +/- 0.60-fold change, test vs controls; p = 0.001) were also observed in the lateral territory of test animals versus controls. Reconstitution of the proximally occluded circumflex artery from patch collaterals was demonstrated on gastroepiploic arteriography in a subset of test animals. CONCLUSIONS: This model results in an angiogenic process of significantly greater magnitude than that resulting from chronic myocardial ischemia alone, without the need for exogenous angiogenic agents.