Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells

Endothelial nitric oxide synthase (eNOS) is essential for neovascularization. Here we show that the impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. Mice deficient in eNOS (Nos3(-/-)) show reduced vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increased mortality after myelosuppression. Intravenous infusion of wild-type progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization of Nos3(-/-) mice in a model of hind-limb ischemia, suggesting that progenitor mobilization from the bone marrow is impaired in Nos3(-/-) mice. Mechanistically, matrix metalloproteinase-9 (MMP-9), which is required for stem cell mobilization, was reduced in the bone marrow of Nos3(-/-) mice. These findings indicate that eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells. This may contribute to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic NO bioactivity.     

Far infra-red therapy promotes ischemia-induced angiogenesis in diabetic mice and restores high glucose-suppressed endothelial progenitor cell functions

ABSTRACT: BACKGROUND: Far infra-red (IFR) therapy was shown to exert beneficial effects in cardiovascular system, but effects of IFR on endothelial progenitor cell (EPC) and EPC-related vasculogenesis remain unclear. We hypothesized that IFR radiation can restore blood flow recovery in ischemic hindlimb in diabetic mice by enhancement of EPCs functions and homing process.Materials and methodsStarting at 4 weeks after the onset of diabetes, unilateral hindlimb ischemia was induced in streptozotocine (STZ)-induced diabetic mice, which were divided into control and IFR therapy groups (n = 6 per group). The latter mice were placed in an IFR dry sauna at 34[DEGREE SIGN]C for 30 min once per day for 5 weeks. RESULTS: Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio in the thermal therapy group was significantly increased beyond that in controls, and significantly greater capillary density was seen in the IFR therapy group. Flow cytometry analysis showed impaired EPCs (Sca-1+/Flk-1+) mobilization after ischemia surgery in diabetic mice with or without IFR therapy (n = 6 per group). However, as compared to those in the control group, bone marrow-derived EPCs differentiated into endothelial cells defined as GFP+/CD31+ double-positive cells were significantly increased in ischemic tissue around the vessels in diabetic mice that received IFR radiation. In in-vitro studies, cultured EPCs treated with IFR radiation markedly augmented high glucose-impaired EPC functions, inhibited high glucose-induced EPC senescence and reduced H2O2 production. Nude mice received human EPCs treated with IFR in high glucose medium showed a significant improvement in blood flow recovery in ischemic limb compared to those without IFR therapy. IFR therapy promoted blood flow recovery and new vessel formation in STZ-induced diabetic mice. CONCLUSIONS: Administration of IFR therapy promoted collateral flow recovery and new vessel formation in STZ-induced diabetic mice, and these beneficial effects may derive from enhancement of EPC functions and homing process.     

Endothelial progenitor cells for regeneration

Endothelial progenitor cells (EPCs) have been recently isolated from peripheral blood and bone marrow (BM), and shown to be incorporated into sites of physiological and pathological neovascularization in vivo. In contrast to differentiated endothelial cells (ECs), transplantation of EPCs successfully enhanced vascular development by in situ differentiation and proliferation within ischemic organs. Based on such a novel concept of closed up function on EPCs in postnatal neovascularization, the beneficial property of EPC is attractive for cell therapy as well as cell-mediated gene therapy applications targeting regeneration of ischemic tissue.     

Corneal endothelial cell density decreases with age in emmetropic eyes

PURPOSE: To analyze the corneal endothelial cell density in healthy adult emmetropic subjects. METHODS: We analyzed the corneal endothelial cell density of a group made up of 225 emmetropic subjects (n=225). As age-matched control groups we analyzed two other groups, one made up of myopic subjects (n=209) and the other made up of hyperopic subjects (n=203). We recorded the mean of three consecutive measurements of the corneal endothelial cell density using the Topcon SP-2000P non-contact specular microscope (Topcon Corp., Tokyo, Japan). RESULTS: The mean age was 38.6+/-11.8 years, 40.7+/-12.2 years, and 39.2+/-10.5 years for emmetropic, myopic and hyperopic subjects respectively (p=0.994). No significant differences (p=0.920) in endothelial cell density values were found between emmetropic (2985+/-245 cells/mm2), myopic (2936+/-258 cells/mm2) and hyperopic eyes (2946+/-253 cells/mm2). Lower corneal endothelial cell density values were found in older emmetropic (p<0.001), myopic (p<0.001), and hyperopic subjects (p<0.001). A significant correlation between endothelial cell density and age was found in emmetropic (r=-0.958; p<0.001), myopic (r= -0.954; p<0.001) and hyperopic subjects (r= -0.948; p<0.001). CONCLUSIONS: In healthy emmetropic subjects there is a reduction in corneal endothelial cell density with age although there are no differences in corneal endothelial cell density values between emmetropic, myopic and hyperopic subjects.     

Direct comparison of human mesenchymal stem cells derived from adipose tissues and bone marrow in mediating neovascularization in response to vascular ischemia.

BACKGROUND/AIM: Although transplantation of MSC derived from bone marrow or adipose tissues has been shown in proangiogenic action in hindlimb ischemia model of nude mice, little information is available regarding comparison of the angiogenic potency between human adipose stromal cells (hADSC) and bone marrow stromal cells (hBMSC). We compared their therapeutic potential by transplantation of equal numbers of hADSC or hBMSC in a nude mice model of hindlimb ischemia. METHODS AND RESULTS: One day after creating hindlimb ischemia, mice were randomized to receive hADSC transplantation (hADSC group), hBMSC transplantation (hBMSC group), or vehicle transplantation (Control group). Two weeks after transplantation, the laser Doppler perfusion index was significantly higher in the hADSC group and hBMSC group than in the control group. Comparison between hADSC and hBMSC group showed better recovery of blood flow in hADSC group than in hBMSC group. Conditioned media from hADSC (hADSC-CM) showed better in vitro tube formation of hADSC than conditioned media from hBMSC (hBMSC-CM). hADSC showed higher expression of MMP3 and MMP9 than hBMSC. A MMP inhibitor, GM6001, and the transfection of MMP3 or MMP9 siRNA oligonucleotides inhibited in vitro tube formation of hADSC. Transplantation of MMP3 or MMP9 siRNA oligonucleotieds-transfected hADSC showed lower blood flow recovery and higher tissue injury than control oligonucelotide-transfected cells. CONCLUSION: This study showed that hADSC can be an ideal source for therapeutic angiogenesis in ischemic disease in terms of efficacy, accessibility and available tissue amounts.     

Improved healing of microvascular PTFE prostheses by induction of a clot layer: an experimental study in rats

This study was undertaken to test the hypothesis that the induction of a clot layer on the graft surface of microvascular polytetrafluoroethylene (PTFE) prostheses might improve their healing. PTFE microvascular prostheses (n = 18), mechanically roughened PTFE microvascular prostheses (n = 18), and Chitosan-impregnated PTFE microvascular prostheses (n = 18) (all prostheses: length 1 cm, inside diameter 1.5 mm, fibril length 30 microns) were implanted into the abdominal aortas of rats and were evaluated at 3 days (n = 3), 10 days (n = 3), 3 weeks (n = 6), and 6 weeks (n = 6) with regard to the presence or absence of a clot layer and with regard to the amount of graft healing. All untreated PTFE prostheses were never found to be covered with a clot layer, only scarcely with some platelets, and showed poor neoendothelial healing; even at 6 weeks after implantation, there was only endothelial cell coverage near the anastomotic sides (coverage = 19 +/- 4 percent). The endothelial cells were present directly on the graft surface. In contrast, both the roughened and the Chitosan-impregnated PTFE prostheses were completely covered with a thin clot layer upon implantation and demonstrated significantly better neoendothelial healing (endothelial cell coverage at 6 weeks = 76 +/- 22 percent and 75 +/- 18 percent, respectively; p less than 0.001); moreover, in these prostheses, the endothelial cells were present on a matrix of smooth-muscle cells, which covered the graft surface completely. These results confirm our hypothesis that the induction of a clot layer on the graft surface of microvascular PTFE prostheses improves their healing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of ultrastructural changes of endothelial cells and astrocytes occurring during blood brain barrier damage after traumatic brain injury with biochemical markers of BBB leakage and inflammatory response

Focal cerebral contusion can be dynamic and expansive. It has been proved that subsequent expansive contusion is caused by brain parenchyma damage, especially BBB damage. We investigated a group of patients with traumatic brain injury. The patients (n=18) were divided into group I (n=7) of patients submitted to neurosurgery due to expansive contusion, and group II (n=11) of patients without surgery. Serum concentrations of NSE and S-100B protein were measured by electrochemiluminescence immunoassay, interleukin-6 (IL-6) was measured by chemiluminescent sequential immunometric assay and matrix metalloproteinases (MMP-9, MMP-2) were measured by immunoassays. Cortical biopsy specimens of brain were investigated by electron microscopy in patients with trauma brain injury submitted to neurosurgery. Biochemical investigation from first day up to third day after traumatic brain injury proved increased values of IL-6 (302.2+/-119.9 vs. 59.6+/-11.9 ng/l, p<0.02) and S-100B protein (3.064+/-1.064 vs. 0.649+/-0.182 microg/l, p<0.05) in patients with expansive lesion compared to patients without expansive contusion. Significantly higher levels of MMP-9 (150.4+/-28.46 vs. 74.11+/-13.16 ng/l, p<0.05) and of MMP-2 (814.5+/-126.3 vs. 523.1+/-25.28 ng/l, p<0.05) were found during first 3 days after admission in group I compared to group II. MMP-9 has also elevated in group II from lower values after admission (74.11+/-13.16 ng/l) up to high levels on the 10th day of hospitalization (225.1+/-49.35 ng/l). Ultrastructural investigation of endothelial cells and surrounded tissue revealed perivascular hemorrhage, increased pinocytic activity of endothelial cells, and cytotoxic edema of astroglial cells. Multivesical bodies were disclosed inside the endothelial cells. Higher levels of serum protein S-100B and IL-6 correlated with ultrastructural changes of endothelial cells, and with inflammatory response following TBI, respectively.     

Human Cerebral Microvessel Endothelial Cell Culture as a Model System to Study the Blood–Brain Interface in Ischemic/Hypoxic Conditions

Summary 1. Cerebral ischemia and reperfusion induce several changes on the endothelial cells at the microcirculatory level.2. Vasogenic brain edema due to compromised blood–brain barrier, transformation of the endothelial cell surface from an anticoagulant to a procoagulant property are important factors in the pathogenesis of ischemic stroke.3. Release of prostaglandins, endothelin-1, complement proteins, and matrix metalloproteinase-9 by microvascular endothelial cells are other components in the complex mechanism of brain ischemia/hypoxia.4. Ultrastructural studies documented the opened paracellular avenues in the course of vasogenic edema in different experimental models.5. Tight junctions of endothelial cells have been characterized with freeze fracture electron microscopy, and the process of transvesiculation was analyzed using rapid freeze and freeze substitution procedure before electron microscopy studies.6. In endothelial cell-culture experiments, we used rodent and later human brains.7. Endothelial cells co-cultured with astroglia resulted in an elaborate tight junctional complex.8. This co-culture technique becomes the basis of in vitro blood–brain barrier studies. On endothelial cells of human brain origin, different regulatory factors found to be responsible for the complex mechanism of ischemic stroke.This paper is dedicated to the memory of F. Joó, the good friend and pioneer in endothelial cell research.This revised article was published online in May 2005 with a February 2005 cover date.     

Rosuvastatin reduces experimental left ventricular infarct size after ischemia-reperfusion injury but not total coronary occlusion

This study compared the effects of rosuvastatin on left ventricular infarct size in mice after permanent coronary occlusion vs. 60 min of ischemia followed by 24 h of reperfusion. Statins can inhibit neutrophil adhesion, increase nitric oxide synthase (NOS) expression, and mobilize progenitor stem cells after ischemic injury. Mice received blinded and randomized administration of rosuvastatin (20 mg.kg(-1).day(-1)) or saline from 2 days before surgery until death. After 60 min of ischemia with reperfusion, infarct size was reduced by 18% (P = 0.03) in mice randomized to receive rosuvastatin (n = 18) vs. saline (n = 22) but was similar after permanent occlusion in rosuvastatin (n = 17) and saline (n = 20) groups (P = not significant). Myocardial infarct size after permanent left anterior descending coronary artery occlusion (n = 6) tended to be greater in NOS3-deficient mice than in the wild-type saline group (33 +/- 4 vs. 23 +/- 2%, P = 0.08). Infarct size in NOS3-deficient mice was not modified by treatment with rosuvastatin (34 +/- 5%, n = 6, P = not significant vs. NOS3-deficient saline group). After 60 min of ischemia-reperfusion, neutrophil infiltration was similar in rosuvastatin and saline groups as was the percentage of CD34(+), Sca-1(+), and c-Kit(+) cells. Left ventricular NOS3 mRNA and protein levels were unchanged by rosuvastatin. Rosuvastatin reduces infarct size after 60 min of ischemia-reperfusion but not after permanent coronary occlusion, suggesting a potential anti-inflammatory effect. Although we were unable to demonstrate that the myocardial protection was due to an effect on neutrophil infiltration, stem cell mobilization, or induction of NOS3, these data suggest that rosuvastatin may be particularly beneficial in myocardial protection after ischemia-reperfusion injury.     

Transplantation of endothelial progenitor cells for therapeutic neovascularization

Endothelial progenitor cells (EPCs), which were first identified in adult peripheral blood mononuclear cells (MNCs), play an important role in postnatal neovascularization. Tissue ischemia augments mobilization of EPCs from bone marrow into the circulation and enhances incorporation of EPCs at sites of neovascularization. Two methods to obtain EPCs from bone marrow, peripheral blood or cord blood MNCs have been evaluated for therapeutic neovascularization: (1) fresh isolation using anti-CD34, anti-KDR or anti-AC133 antibody, and (2) ex vivo expansion of total MNCs. In an immunodeficient mouse model of hindlimb ischemia, systemic transplantation of human ex vivo expanded EPCs improves limb survival through the enhancement of blood flow in the ischemic tissue. A similar strategy also leads to histological and functional preservation of ischemic myocardium of nude rats. Recently, a preclinical study of catheter-based, intramyocardial transplantation ofautologous EPCs in a swine model of chronic myocardial ischemia demonstrated the therapeutic potential of cell-based therapy, with attenuation of myocardial ischemia and improvement in left ventricular function. These favorable outcomes strongly suggest a therapeutic impact of EPC transplantation in clinical settings. Further basic research, with improved understanding of the mechanisms governing homing and incorporation of EPCs, will be still necessary to optimize the methodology of the cell therapy.     

Augmentation of Neovascularizaiton in Hindlimb Ischemia by Combined Transplantation of Human Embryonic Stem Cells-Derived Endothelial and Mural Cells

Background

We demonstrated that mouse embryonic stem (ES) cells-derived vascular endothelial growth factor receptor-2 (VEGF-R2) positive cells could differentiate into both endothelial cells (EC) and mural cells (MC), and termed them as vascular progenitor cells (VPC). Recently, we have established a method to expand monkey and human ES cells-derived VPC with the proper differentiation stage in a large quantity. Here we investigated the therapeutic potential of human VPC-derived EC and MC for vascular regeneration.

Methods and Results

After the expansion of human VPC-derived vascular cells, we transplanted these cells to nude mice with hindlimb ischemia. The blood flow recovery and capillary density in ischemic hindlimbs were significantly improved in human VPC-derived EC-transplanted mice, compared to human peripheral and umbilical cord blood-derived endothelial progenitor cells (pEPC and uEPC) transplanted mice. The combined transplantation of human VPC-derived EC and MC synergistically improved blood flow of ischemic hindlimbs remarkably, compared to the single cell transplantations. Transplanted VPC-derived vascular cells were effectively incorporated into host circulating vessels as EC and MC to maintain long-term vascular integrity.

Conclusions

Our findings suggest that the combined transplantation of human ES cells-derived EC and MC can be used as a new promising strategy for therapeutic vascular regeneration in patients with tissue ischemia.     

Statins enhance clonal growth of late outgrowth endothelial progenitors and increase myocardial capillary density in the chronically ischemic heart

Background

Coronary artery disease and ischemic heart disease are leading causes of heart failure and death. Reduced blood flow to heart tissue leads to decreased heart function and symptoms of heart failure. Therapies to improve heart function in chronic coronary artery disease are important to identify. HMG-CoA reductase inhibitors (statins) are an important therapy for prevention of coronary artery disease, but also have non-cholesterol lowering effects. Our prior work showed that pravastatin improves contractile function in the chronically ischemic heart in pigs. Endothelial progenitor cells are a potential source of new blood vessels in ischemic tissues. While statins are known to increase the number of early outgrowth endothelial progenitor cells, their effects on late outgrowth endothelial progenitor cells (LOEPCs) and capillary density in ischemic heart tissue are not known. We hypothesized that statins exert positive effects on the mobilization and growth of late outgrowth EPCs, and capillary density in ischemic heart tissue.

Methodology/Principal Findings

We determined the effects of statins on the mobilization and growth of late outgrowth endothelial progenitor cells from pigs. We also determined the density of capillaries in myocardial tissue in pigs with chronic myocardial ischemia with or without treatment with pravastatin. Pravastatin therapy resulted in greater than two-fold increase in CD31+ LOEPCs versus untreated animals. Addition of pravastatin or simvastatin to blood mononuclear cells increased the number of LOEPCs greater than three fold in culture. Finally, in animals with chronic myocardial ischemia, pravastatin increased capillary density 46%.

Conclusions

Statins promote the derivation, mobilization, and clonal growth of LOEPCs. Pravastatin therapy in vivo increases myocardial capillary density in chronically ischemic myocardium, providing an in vivo correlate for the effects of statins on LOEPC growth in vitro. Our findings provide evidence that statin therapy can increase the density of capillaries in the chronically ischemic heart.     

Lung Ischemia: A Model for Endothelial Mechanotransduction

Endothelial cells in vivo are constantly exposed to shear associated with blood flow and altered shear stress elicits cellular responses (mechanotransduction). This review describes the role of shear sensors and signal transducers in these events. The major focus is the response to removal of shear as occurs when blood flow is compromised (i.e., ischemia). Pulmonary ischemia studied with the isolated murine lung or flow adapted pulmonary microvascular endothelial cells in vitro results in endothelial generation of reactive oxygen species (ROS) and NO. The response requires caveolae and is initiated by endothelial cell depolarization via K_ATP channel closure followed by activation of NADPH oxidase (NOX2) and NO synthase (eNOS), signaling through MAP kinases, and endothelial cell proliferation. These physiological mediators can promote vasodilation and angiogenesis as compensation for decreased tissue perfusion.     

Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells

Tissue engineering may offer patients new options when replacement or repair of an organ is needed. However, most tissues will require a microvascular network to supply oxygen and nutrients. One strategy for creating a microvascular network would be promotion of vasculogenesis in situ by seeding vascular progenitor cells within the biopolymeric construct. To pursue this strategy, we isolated CD34(+)/CD133(+) endothelial progenitor cells (EPC) from human umbilical cord blood and expanded the cells ex vivo as EPC-derived endothelial cells (EC). The EPC lost expression of the stem cell marker CD133 but continued to express the endothelial markers KDR/VEGF-R2, VE-cadherin, CD31, von Willebrand factor, and E-selectin. The cells were also shown to mediate calcium-dependent adhesion of HL-60 cells, a human promyelocytic leukemia cell line, providing evidence for a proinflammatory endothelial phenotype. The EPC-derived EC maintained this endothelial phenotype when expanded in roller bottles and subsequently seeded on polyglycolic acid-poly-l-lactic acid (PGA-PLLA) scaffolds, but microvessel formation was not observed. In contrast, EPC-derived EC seeded with human smooth muscle cells formed capillary-like structures throughout the scaffold (76.5 +/- 35 microvessels/mm(2)). These results indicate that 1) EPC-derived EC can be expanded in vitro and seeded on biodegradable scaffolds with preservation of endothelial phenotype and 2) EPC-derived EC seeded with human smooth muscle cells form microvessels on porous PGA-PLLA scaffolds. These properties indicate that EPC may be well suited for creating microvascular networks within tissue-engineered constructs.     

骨髓间充质干细胞和内皮祖细胞移植促进血流重建的比较

目的:比较骨髓间充质细胞(Bone Marrow Mesenchymal Stem Cells,BM/MSC)和骨髓源内皮祖细胞(Bone Marrow Endothelialprogenitor cells,BM/EPC)移植促进血流重建的效果,为进一步优化骨髓干细胞移植治疗肢体缺血提供理论基础。方法:获取Lewis大鼠骨髓单个核细胞,在体外培养分化为MSC和EPC。采用Lewis大鼠建立单侧后肢缺血模型。在模型建立后3天,将0.8mlD-Hanks液注入大鼠缺血侧后肢,为对照组(n=6);将8×106个骨髓MSC植入大鼠缺血侧后肢,为MSC组(n=6);将体外培养的8×106个EPC植入大鼠缺血侧后肢,为EPC组(n=6)。细胞移植后3周行缺血大鼠后肢动脉造影,检测缺血侧后肢侧支血管数;获取缺血侧后肢腓肠肌,分别行CD31和α-SMA免疫组化染色,计算毛细血管密度和小动脉密度。结果:MSC组与EPC组侧支血管数无显著性差异,二者均高于对照组;EPC组毛细血管密度明显高于MSC组,二者均高于对照组;MSC组与EPC组小动脉密度无显著性差异,二者均高于对照组。结论:骨髓间充质干细胞移植和内皮祖细胞移植均能够明显促进血流重建,而且骨髓间充质干细胞在治疗肢体缺血性疾病中的优势应该受到重视。     

Attenuation of ischemic preconditioning in pigs by scavenging of free oxyradicals with ascorbic acid

Free oxyradicals are involved in the signal transduction of ischemic preconditioning in rats and rabbits. Data from larger mammals in which the infarct development is closer to that in humans are lacking. We have therefore investigated the impact of the radical scavenger ascorbic acid on ischemic preconditioning in pigs. In 33 anesthetized pigs, the left anterior descending coronary artery was perfused from an extracorporeal circuit. Infarct size (measured as percent area at risk) was determined by triphenyltetrazolium chloride staining. In placebo-treated animals undergoing 90 min of severe ischemia and 120 min of reperfusion, infarct size averaged 26.9 +/- 3.9% (mean +/- SE; n = 9). Ischemic preconditioning by 10 min of ischemia and 15 min of reperfusion reduced infarct size to 6.4 +/- 2.4% (P < 0.05 vs. placebo; n = 9). Intravenous infusion of ascorbic acid (30 min before ischemic preconditioning or ischemia; 2-g bolus followed by 25 mg/min until the end of ischemia) had no effect on infarct size per se (22.6 +/- 6.5%; n = 6), but largely abolished the infarct size reduction by ischemic preconditioning (19.1 +/- 5.4%; n = 9). Scavenging of free oxyradicals with ascorbic acid largely attenuates the beneficial effect of ischemic preconditioning in pigs.     

内皮祖细胞的生物学特性及其临床应用前景

内皮祖细胞（Endothelial Progenitor Cells,EPCs）是内皮细胞（endothelial cells,ECs）的前体细胞,即能分化为成熟ECs的祖细胞,它在血管内皮再生中发挥着重要作用。随着EPCs研究的深入,其在临床诊断、预后判断和各种缺血性疾病的治疗方面将会有广阔的应用前景。然而,关于EPCs的定义、来源、表面标记以及培养鉴定方法目前仍存在争议。     

Digital image analysis of cytoskeletal F-actin disintegration in renal microvascular endothelium following ischemia/reperfusion.

BACKGROUND: Damaged and/or dysfunctional microvascular endothelium has been implicated in the pathogenesis of ischemic acute renal failure (ARF). Rapidly occurring changes in the endothelial F-actin cytoskeleton as observed in vitro might be responsible, but have been proven difficult to measure accurately in situ. Therefore, the purpose of this study was to examine several methods of digital image analysis in order to quantify the alterations of endothelial F-actin after renal ischemia and reperfusion (I/R), and to relate these to deterioration of renal function. METHODS: Frozen sections of Sham and I/R rat kidneys were fixed in 4% formaldehyde and stained with rhodamine-phallo?din. Microvascular structures were captured using a 3i Marianastrade mark digital imaging fluorescence microscope workstation. Images were analyzed using 3i SlideBooktrade mark software, employing several masking techniques and line-scans. RESULTS: Digital image analysis demonstrated a decrease in the mean intensity of rhodamine-phallo?din fluorescence after I/R from 1030 +/- 187 to 735 +/- 121 a.u. (arbitrary units, mean +/- SD, n = 7). The number of F-actin fragments per pixel increased from (15.8 +/- 4.9) x 10(-5) to (20.7 +/- 3.5) x 10(-5) (n = 7), indicating cytoskeletal fragmentation. In addition, line-scan analysis demonstrated a disturbed spatial orientation of the F-actin cytoskeleton after I/R. Finally, the loss of F-actin correlated with a rise in plasma creatinine. CONCLUSIONS: The methods of digital image analysis described in the present study demonstrate that renal I/R induces profound changes in the F-actin cytoskeletal structure of microvascular endothelial cells, implicating an injured and dysfunctional microvascular endothelium, which may contribute to acute renal failure (ARF).