In vivo assessment of acute microvascular injury after reperfusion of ischemic tibialis anterior muscle of the hamster

The purposes of this study were to develope an in vivo model of skeletal muscle ischemia-reperfusion to assess the patterns of microvascular injury, to evaluate a scoring system that permits quantitation of this injury, and to determine in vivo the extent of white blood cell adhesion within the microcirculation during the acute postreperfusion period. Syrian golden hamsters underwent 3.0 or 4.5 hr of lower extremity ischemia without anticoagulation. The microcirculation of the tibialis anterior muscle was visualized by fluorescent intravital microscopy (700X). During the first 1.5 hr of reperfusion the microvascular injury was scored by a grading system based upon the extent of extravasation of fluorosceinlabeled albumin and the degree and level of microvessel obstruction. To correlate the observed changes in the microcirculation to changes in the whole muscle, in a separate group of animals, pH changes in the tibialis anterior muscle were measured at the same time intervals under identical experimental conditions as the microvascular measurements. White blood cells were transiently fluoresced at 1.5 hr after reperfusion by intravenous acridine red and the number of white blood cells rolling (rollers) or sticking (stickers) to the endothelium during a 30-sec observation period was recorded. Two distinct patterns of microvascular injury were seen: after 3.0 hr of ischemia there was a progressive extravasation, some capillary but no arteriolar or venular obstruction, flow velocities increased over time; after 4.5 hr of ischemia there was a greater heterogeneity of injury, primary "no reflow", extensive capillary, arteriolar, and venular obstruction, as well as a progressive decline in flow velocities. Thrombosis of microvessels was rare. There was no inflow vessel thrombosis. The scoring system showed significant differences, mean (SE), in both the extent of early (30 min) and late (90 min) injuries for the 3.0-hr (early 1.4 (0.6) vs late 2.0 (0.5)) and the 4.5-hr periods of ischemia (early 2.3 (0.9) vs late 3.0 (0.8)). In addition, pH measurements of the tibialis anterior paralleled the changes observed in the microcirculation. There was a significant increase in the number of rolling WBCs, 14.3 (10.3), in the animals undergoing 3.0 hr of ischemia, and in both the number of rollers, 7.4 (4.3), and stickers, 4.0 (1.9), in the animals undergoing 4.5 hr of ischemia compared to those of controls, 1.7 (2.4) and 1.9 (1.5). These results are the first in vivo observations of the pattern of microvascular injury and the extent of white blood cell adhesion in a true muscle of locomotion. We believe that this model will allow further clarification of the mechanisms responsible for the microvascular injury that occurs after reperfusion of ischemic skeletal muscle.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28552/1/0000351.pd

Type 2 Diabetes-Induced Hematopoietic Stem Cell Oxidant Stress Attenuates the Differentiation, Skews M1/M2 Specification of Monocytes/Macrophages and Delays Wound Healing in db/db Mice

Rationale: After recruitment to wounds, monocytes differentiate into macrophages which play a central role in all stages of wound healing. Wound healing is significantly delayed in type 2 diabetics. Although accumulating evidence suggests that delayed wound healing in type 2 diabetics is related to macrophages specification into M1/M2 phenotypes, the mechanism remains unknown. Objective: This study tested the hypothesis that type 2 diabetes induces hematopoietic stem cells (HSCs) oxidant stress that reduces their differentiation towards monocytes and skews the specification of M1/M2 phenotype, thereby causing delayed wound healing. Methods and Results: HSCs were sorted from bone marrow of WT and db/db type 2 diabetic mice. DCF staining showed significant oxidant accumulation in HSCs from db/db mice which was reversed by the antioxidant, N-acetylcysteine (NAC). Bone marrow monocyte concentration (FACS analysis of cell surface markers f4/80, cd14 and cd115) was significantly lower in db/db mice than in WT mice. NAC also reversed the reduced differentiation towards monocytes. Wound closure rate was significantly delayed in db/db mice. Macrophages were isolated from wounds and their concentration and M1/M2 phenotype were quantified by flow cytometry. During the inflammatory phase of wound healing, macrophage concentration was decreased and the proportion of M1 macrophages was lower in db/db mice than in WT mice. During new tissue formation phase, macrophage concentration was decreased and the proportion of M2 macrophage was lower, but M1 macrophage was higher in db/db mice than in WT mice. During tissue remodeling phase, macrophage concentration was increased and M1 macrophage remained higher in db/db mice, but no difference was observed in the proportion of M2 macrophages. The reduced differentiation of HSCs towards monocytes and the delayed wound closure phenotype of db/db mice could be transferred to WT mice by transplanting db/db HSCs into lethally irradiated WT mice. Conclusion: Type 2 diabetes-induced HSC oxidant stress impairs HSC differentiation towards monocytes, skews the M1/M2 specification of macrophages and thereby accounts for the delayed wound healing. Type 2 diabetes-induced HSC oxidant stress may be a heretofore unrecognized critical regulator of dysinflammation in type 2 diabetic

Effect of adenoviral titer and instillation pressure on gene transfer efficiency to arterial and venous grafts ex-vivo

AbstractObjective: Adenoviral-mediated gene transfer to arterial and venous grafts has potential in the treatment of a number of vascular diseases. Despite widespread use of these vectors to mediate gene transfer to blood vessel walls, the optimal transduction conditions for each type of vessel has yet to be determined. Our objective was to study the effect of adenoviral titer and instillation pressure on efficiency of gene transfer to arterial and venous grafts ex-vivo. Methods: Jugular vein and carotid artery segments of 8 cm were harvested from Yorkshire Cross pigs. Tissue culture media or different titers of an adenoviral vector encoding human placental alkaline phosphatase (hpAP) were instilled into venous and arterial grafts at 0 mm Hg or 80 to 100 mm Hg of pressure and bathed externally in the same solution at 37° C for 30 minutes. The grafts were rinsed, opened longitudinally, and incubated in culture media at 37° C for 48 hours. Grafts were fixed and stained for hpAP transgene expression to quantitate percent luminal transduction or homogenized for alkaline phosphatase (AP) activity to determine total transmural transduction. Results: For venous grafts, the percent luminal area stained for hpAP was greatest with 108 plaque-forming units/mL at 0 mm Hg (81% ± 7%) and decreased with increasing titers (53% ± 9% at 109 pfu/mL and 44% ± 11% at 5 × 109 pfu/mL; n = 7; P < .05). No increase in percent luminal area stain was achieved with an instillation pressure of 80 to 100 mm Hg at any viral titer. The inverse finding was observed in arterial grafts. For arterial grafts, the greatest percent luminal area stained was achieved with 5 × 109 pfu/mL at 80 to 100 mm Hg (76% ± 7%). An instillation pressure of 80 to 100 mm Hg increased the percent luminal area stained at 108 pfu/mL from 31% ± 9% to 66% ± 8% (n = 8; P = .01). For venous grafts, total AP activity peaked with 109 pfu/mL at 0 mm Hg and decreased with an instillation pressure of 80 to 100 mm Hg (30.6 ± 9.7 U/mg versus 10.9 ± 2.5 U/mg; n = 7; P < .01). However, for arterial grafts, total AP activity peaked with 5 × 109 pfu/mL (0 mm Hg) and increased with an instillation pressure of 80 to 100 mm Hg (32.8 ± 9.9 U/mg versus 63.4 ± 20.5 U/mg; n = 8; P < .05). Conclusion: High transduction efficiency can be achieved with adenoviral-mediated gene transfer of arterial and venous grafts. Gene transfer with the vascular graft's physiologic pressure conditions improved transduction efficiency for the artery (80 to 100 mm Hg) and vein (0 mm Hg). Comprehensive analysis of adenoviral transduction conditions is important to realize the full promise of adenoviral-mediated gene transfer. (J Vasc Surg 2002;36:263-70.

Effect of calcium on the vasoactive response of arterioles to light during fluorescent intravital microscopy of the tibialis anterior muscle of the hamster

Arterioles may undergo a transient vasoactive response when exposed to light during fluorescent intravital microscopy. We hypothesized that the type and frequency of the vasoactive responses by arterioles to light is calcium dependent. In order to test this hypothesis we quantitated the type and frequency of vasoactive responses by arterioles to light in the presence and absence of calcium within the suffusate bathing the muscle. In addition, we determined whether the presence or absence of calcium also influenced the reactivity of these arterioles to adenosine and norepinephrine. In separate experiments, we determined the effect of increases in suffusate calcium concentration on the resting diameter of arterioles. The concentration of calcium in the suffusate significantly influenced the frequency of light-induced vasoactivity of the arterioles of the tibialis anterior muscle: a normal suffusate calcium concentration of 2 x 10-3M was associated with an incidence of light-induced vasoreactivity of 77% but decreased significantly to 58% when calcium was removed from the suffusate. Although the frequency of the vasoactive response to light was different for the two experimental conditions, the type of vasoactive response, predominately vasomotion, was similar. The vasoactive responses of arterioles to adenosine and norepinephrine were similar in the presence and absence of calcium in the suffusate. However, the concentration of calcium in the suffusate did significantly influence resting arteriolar diameters: 2 x 10-3M calcium CaCl2 caused a mean decrease in the arteriolar diameter of 11.6 (+/-3.0)%, 4 x 10-3M CaCl2 caused a mean decrease of 41.2 (+/-12)%, and 8 x 10-3M CaCl2 caused a mean decrease of 55.4 (+/-14.4)%. These results show that the concentration of calcium in the suffusate bathing the tibialis anterior muscle during fluorescent intravital microscopy significantly influences the vasoactive response of arterioles to light. Further investigation of the mechanisms of light-induced effects on microcirculation during fluorescent intravital microscopy will become important as this technique is used more widely in the study of the microcirculation of solid tissues and organs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29753/1/0000091.pd

Hypercholesterolemia Accelerates the Aging Phenotypes of Hematopoietic Stem Cells by a Tet1-Dependent Pathway

Hypercholesterolemia accelerates the phenotypes of aging in hematopoietic stem cells (HSCs). As yet, little is known about the underlying mechanism. We found that hypercholesterolemia downregulates Ten eleven translocation 1 (Tet1) in HSCs. The total HSC population was increased, while the long-term (LT) population, side population and reconstitution capacity of HSCs were significantly decreased in Tet1(-/-) mice. Expression of the Tet1 catalytic domain in HSCs effectively restored the LT population and reconstitution capacity of HSCs isolated from Tet1(-/-) mice. While Tet1 deficiency upregulated the expression of p19 and p21 in HSCs by decreasing the H3K27me3 modification, the restoration of Tet1 activity reduced the expression of p19, p21 and p27 by restoring the H3K27me3 and H3K36me3 modifications on these genes. These results indicate that Tet1 plays a critical role in maintaining the quiescence and reconstitution capacity of HSCs and that hypercholesterolemia accelerates HSC aging phenotypes by decreasing Tet1 expression in HSCs

The effect of gradual or acute arterial occlusion on skeletal muscle blood flow, arteriogenesis, and inflammation in rat hindlimb ischemia

BackgroundCurrent experimental models of critical limb ischemia are based on acute ischemia rather than on chronic ischemia. Human peripheral vascular disease is largely a result of chromic ischemia. We hypothesized that a model of chronic hindlimb ischemia would develop more collateral arteries, more blood flow, and less necrosis and inflammation than would acute hindlimb ischemia. We therefore developed a rat model of chronic hindlimb ischemia and compared the effects of chronic ischemia with those of acute ischemia on hindlimb skeletal muscle.MethodsAcute or chronic ischemia was induced in 36 male Sprague-Dawley rats. Chronic ischemia caused blood flow, as measured by laser Doppler scanning and confirmed by muscle oxygen tension measurements, to gradually decrease over 1 to 2 weeks after operation.ResultsHistologic analysis showed chronic hindlimb ischemia better preserved muscle mass and architecture and stimulated capillary angiogenesis, while lacking the muscle necrosis and inflammatory cell infiltrate seen after acute ischemia. Surprisingly, the chronic ischemia group recovered dermal blood flow more slowly and less completely than did the acute ischemia group, as measured by laser Doppler (0.66 ± 0.02 vs 0.76 ± 0.04, P < .05) and tissue oxygen tension (0.61 ± 0.06 vs 0.81 ± 0.05, P < .05) at 40 days postoperatively. Consistent with poorer blood flow recovery, chronic ischemia resulted in smaller diameter collateral arteries (average diameter of the five largest collaterals on angiogram was 0.01 ± 0.0003 mm vs 0.013 ± 0.0007 mm for acute, P < .005 at 40 days postoperatively). Acute ischemia resulted in decreased tissue concentrations of vascular endothelial growth factor (VEGF) (0.96 ± 0.23 pg/mg of muscle for acute vs 4.4 ± 0.75 and 4.8 ± 0.75 pg/mg of muscle for unoperated and chronic, respectively, P < .05 acute vs unoperated), and in increased tissue concentrations of interleukin (IL)-1β (7.3 ± 4.0 pg/mg of muscle for acute vs undetectable and 1.7 ± 1.6pg/mg of muscle for unoperated and chronic, respectively, P < 0.05 acute vs unoperated).ConclusionsWe describe here the first model of chronic hindlimb ischemia in the rat. Restoration of blood flow after induction of hindlimb ischemia is dependent on the rate of arterial occlusion. This difference in blood flow recovery correlates with distinct patterns of muscle necrosis, inflammatory cell infiltration, and cytokine induction in the ischemic muscle. Differences between models of acute and chronic hindlimb ischemia may have important consequences for future studies of mechanisms regulating arteriogenesis and for therapeutic approaches aimed at promoting arteriogenesis in humans suffering from critical limb ischemia.Clinical relevanceDespite the substantial clinical differences between acute and chronic ischemia, researchers attempting to develop molecular therapies to treat critical limb ischemia have only tested those therapies in experimental models of acute hindlimb ischemia. We present here a novel model of chronic hindlimb ischemia in the rat. We further demonstrate that when hindlimb ischemia is developed chronically, collateral artery development is poorer than when hindlimb ischemia is developed acutely. These findings suggest that further tests of molecular therapies for critical limb ischemia should be performed in chronic hindlimb ischemia models rather than in acute hindlimb ischemia models

Overexpression of endothelial nitric oxide synthase increases skeletal muscle blood flow and oxygenation in severe rat hind limb ischemia

AbstractObjectiveAlthough nitric oxide (NO) has a critical role in angiogenesis, the therapeutic potential of NO synthase overexpression in severe ischemia remains undefined. We tested the hypothesis that overexpression of endothelial NO synthase (eNOS) would improve tissue perfusion in severe hind limb ischemia.MethodsSevere hind limb ischemia was induced in 122 adult male Sprague-Dawley rats. Ten days after the induction of hind limb ischemia, vascular isolation and intraarterial delivery of an adenoviral vector encoding eNOS (AdeNOS), a control adenoviral vector (AdE1), or phosphate-buffered saline solution (PBS) was performed. Skeletal muscle blood flow, muscle oxygen tension, angiography, and immunohistochemistry for capillary counts were measured.ResultsGene transfer of AdeNOS increased eNOS protein expression and enzyme activity. Two weeks after gene transfer, skeletal muscle blood flow was fourfold higher in eNOS-transduced than in AdE1-transduced or PBS treated rats and was similar to exercise-induced maximal flow in nonischemic muscle. eNOS overexpression increased muscle oxygen tension in a titer-dependent fashion. This increase persisted 1 month after transduction, even though eNOS enzyme activity had declined to normal levels. Angiography and capillary counts showed that eNOS overexpression increased the size and number of collateral arteries, but did not significantly increase the capillary–muscle fiber ratio.ConclusionseNOS overexpression in an ischemic rat hind limb significantly increased skeletal muscle blood flow, muscle oxygen tension, and collateral arteries (arteriogenesis). Furthermore, eNOS overexpression did not result in capillary angiogenesis above control levels. These studies demonstrate the potential for eNOS overexpression as treatment for severe limb ischemia in human beings

Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: Roles of endothelial nitric oxide synthase and endothelial progenitor cells

ObjectiveWe sought to directly compare the effects of type 1 and type 2 diabetes on postischemic neovascularization and evaluate the mechanisms underlying differences between these groups. We tested the hypothesis that type 2 diabetic mice have a greater reduction in endothelial nitric oxide synthase (eNOS) expression, a greater increase in oxidative stress, and reduced arteriogenesis and angiogenesis, resulting in less complete blood flow recovery than type 1 diabetic mice after induction of hind limb ischemia.MethodsHind limb ischemia was generated by femoral artery excision in streptozotocin-treated mice (model of type 1 diabetes), in Leprdb/db mice (model of type 2 diabetes), and in control (C57BL/6) mice. Dependent variables included eNOS expression and markers of arteriogenesis, angiogenesis, and oxidative stress.ResultsPostischemia recovery of hind limb perfusion was significantly less in type 2 than in type 1 diabetic mice; however, neither group demonstrated a significant increase in collateral artery diameter or collateral artery angioscore in the ischemic hind limb. The capillary/myofiber ratio in the gastrocnemius muscle decreased in response to ischemia in control or type 1 diabetic mice but remained the same in type 2 diabetic mice. Gastrocnemius muscle eNOS expression was lower in type 1 and 2 diabetic mice than in control mice. This expression decreased after induction of ischemia in type 2 but not in type 1 diabetic mice. The percentage of endothelial progenitor cells (EPC) in the peripheral blood failed to increase in either diabetic group after induction of ischemia, whereas this variable significantly increased in the control group in response to ischemia. EPC eNOS expression decreased after induction of ischemia in type 1 but not in type 2 diabetic mice. EPC nitrotyrosine accumulation increased after induction of ischemia in type 2 but not in type 1 diabetic mice. EPC migration in response to vascular endothelial growth factor was reduced in type 1 and type 2 diabetic mice vs control mice. EPC incorporation into tubular structures was less effective in type 2 diabetic mice. Extensive fatty infiltration was present in ischemic muscle of type 2 but not in type 1 diabetic mice.ConclusionType 2 diabetic mice displayed a significantly less effective response to hind limb ischemia than type 1 diabetic mice.Clinical RelevanceDiabetes is important in the pathogenesis of peripheral artery disease. The present study demonstrates that the vascular response to acute hind limb ischemia is dependent on the type of diabetes present. Type 2 diabetic mice (Leprdb/db) demonstrated significantly less effective blood flow recovery than type 1 diabetic mice (streptozotocin-induced). Moreover, the differences between diabetic groups appeared contingent, at least in part, on differences in endothelial nitric oxide, oxidant stress, and endothelial progenitor cell function between the two diabetic groups. Although direct extrapolation of animal data to the human experience must be made with caution, these findings indicate that the type of diabetes present, and not just the presence of diabetes per se, may be important in the initiation of progression of peripheral artery disease

Type 2 Diabetes Impairs the Ability of Skeletal Muscle Pericytes to Augment Postischemic Neovascularization in db/db Mice

Peripheral artery disease is an atherosclerotic occlusive disease that causes limb ischemia and has few effective noninterventional treatments. Stem cell therapy is promising, but concomitant diabetes may limit its effectiveness. We evaluated the therapeutic potential of skeletal muscle pericytes to augment postischemic neovascularization in wild-type and type 2 diabetic (T2DM) mice. Wild-type C57BL/6J and leptin receptor spontaneous mutation db/db T2DM mice underwent unilateral femoral artery excision to induce limb ischemia. Twenty-four hours after ischemia induction, CD45-CD34-CD146+ skeletal muscle pericytes or vehicle controls were transplanted into ischemic hindlimb muscles. At postoperative day 28, pericyte transplantation augmented blood flow recovery in wild-type mice (79.3 ± 5% vs. 61.9 ± 5%; P = 0.04), but not in T2DM mice (48.6% vs. 46.3 ± 5%; P = 0.51). Pericyte transplantation augmented collateral artery enlargement in wild-type (26.7 ± 2 µm vs. 22.3 ± 1 µm, P = 0.03), but not T2DM mice (20.4 ± 1.4 µm vs. 18.5 ± 1.2 µm, P = 0.14). Pericyte incorporation into collateral arteries was higher in wild-type than in T2DM mice (P = 0.002). Unexpectedly, pericytes differentiated into Schwann cells in vivo. In vitro, Insulin increased Nox2 expression and decreased tubular formation capacity in human pericytes. These insulin-induced effects were reversed by N-acetylcysteine antioxidant treatment. In conclusion, T2DM impairs the ability of pericytes to augment neovascularization via decreased collateral artery enlargement and impaired engraftment into collateral arteries, potentially via hyperinsulinemia-induced oxidant stress. While pericytes show promise as a unique form of stem cell therapy to increase postischemic neovascularization, characterizing the molecular mechanisms by which T2DM impairs their function is essential to achieve their therapeutic potential

Uncommon splanchnic artery aneurysms: Pancreaticoduodenal, gastroduodenal, superior mesenteric, inferior mesenteric, and colic

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41364/1/10016_2005_Article_BF02000601.pd