Biomedical subjects

W N Durán

Publications and source records attributed to W N Durán.

At least 19 recordsLinked to original sources

PubMed2001

VEGF increases permeability of the endothelial cell monolayer by activation of PKB/akt, endothelial nitric-oxide synthase, and MAP kinase pathways.

VEGF is a key regulator of vascular permeability. However, its signaling pathways are incompletely understood. We tested the hypothesis that VEGF regulates endothelial cell (EC) permeability by activating PKB/akt, NOS, and MAP kinase dependent pathways using human umbilical vein EC (HUVEC). Permeability was measured from FITC-dextran 70-kDa flux across the EC monolayer at baseline and after VEGF at 0.034, 0.068, 1, 10, and 100 nM. VEGF increased HUVEC permeability to FITC-dextran in a dose-dependent manner. VEGF (1 nM) increased permeability from 3.9 x 10(-6) +/- 0.7 x 10(-6) to 14.0 x 10(-6) +/- 1.7 x 10(-6) cm/s (mean +/- SEM; P < 0.001). Permeability changes were also assessed after treatment with 1, 10, and 100 nM wortmannin (PI 3-kinase inhibitor); 0.01, 0.1, and 1.0 nM LY294002 (PI 3-kinase inhibitor); 200 microM l-NMMA (NOS inhibitor); 2.7 microM AG126 (p42/44(MAPK) inhibitor); and 0.006, 0.06, and 0.6 microM SB203580 (p38(MAPK) inhibitor). All inhibitors blocked VEGF-induced permeability changes. Our data demonstrate that (1) VEGF increases permeability of EC monolayers in a dose-dependent fashion, and (2) VEGF-induced permeability is mediated through PI-3 kinase-PKB, NOS, and MAP-kinase signaling cascades. These observations suggest that microvascular hyperpermeability associated with inflammation and vascular disease is mediated by activation of these EC signaling pathways.

Capillary Permeability↗

PubMed2001

Role of matrix metalloproteinases 1, 2, and 9 and tissue inhibitor of matrix metalloproteinase-1 in chronic venous insufficiency.

PURPOSE: Increased transforming growth factor-beta(1) (TGF-beta(1)) activity is associated with chronic venous insufficiency (CVI) disease progression and dermal skin pathology. Because TGF-beta(1) stimulates collagen synthesis and alters the levels of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), we investigated the hypothesis that increased TGF-beta(1) activity is associated with differences in messenger RNA and protein levels of MMPs and TIMP-1 in patients with CVI. METHODS: One hundred ten biopsies of the lower calf and lower thigh in 73 patients were snap frozen in liquid nitrogen and stratified into six groups according to the clinical etiologic anatomic distribution pathophysiology disease classification. One set of lower-calf and lower-thigh biopsies were analyzed for MMP-1 and TIMP-1 gene expression with quantitative reverse transcription and competitive polymerase chain reaction. A second set of biopsies was analyzed for the active and latent forms of MMP-1, MMP-2, and MMP-9 as well as for TIMP-1 by western blotting, gelatin zymography, and tissue localization by immunohistochemistry (IHC). RESULTS: Compared with the control, MMP-1 messenger RNA was increased in class-4 and class-6 patients (P < or =.01), whereas TIMP-1 was increased in class-6 patients only (P < or =.05). However, there were no differences in total protein between MMP-1 and TIMP-1. Active MMP-2 protein increased in class-4 and class-5 patients compared with active MMP-1 and TIMP-1 (P < or =.01). Western blotting did not identify the active component of MMP-9. Similarly, only the latent form of MMP-9 was observed by gelatin zymography, whereas both the latent and active forms of MMP-2 were observed. IHC demonstrated MMP-1 and MMP-2 in dermal fibroblasts and in perivascular leukocytes. TIMP-1 was observed in basal-layer keratinocytes of the epidermis only. MMP-9 was not detected by IHC. CONCLUSION: MMP synthesis is regulated at both the transcriptional and post-transcriptional levels in CVI. Our data suggest that post-translational modifications are key to functional regulation. Dermal fibroblasts and migrating leukocytes are probable cellular sources of MMPs. Increased active MMP-2 levels in class-4 and class-5 patients indicate tissue remodeling caused by pre-ulcer and postulcer environmental stimuli. These data suggest that alterations in MMP-2 activity, in conjunction with TGF-beta(1)-mediated events, cause an imbalance in tissue remodeling leading to a pro-ulcer-forming environment.

Blotting, Western↗

PubMed2000

Platelet-activating factor modulates microvascular dynamics through phospholipase C in the hamster cheek pouch.

We studied the interactions between platelet-activating factor (PAF) and phospholipase C (PLC) in the modulation of microvascular responses in the hamster cheek pouch using intravital microscopy and computer-assisted image analysis. Changes in arteriolar diameter and in integrated optical intensity (IOI, an index of vascular permeability) were measured. Fluorescein-isothiocyanate-labeled dextran 150 (FITC-Dx 150) served as a tracer for macromolecular transport. 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5,-dione (U-73122), two PLC inhibitors, were applied topically in separate experiments. PAF at 10(-7) M elevated IOI from baseline to a mean +/- SEM value of 70. 7 +/- 8.9 units. Pretreatment with 10(-4) and 10(-5) M NCDC and with U-73122 at 10(-5) and 10(-6) M attenuated the maximal increment in mean IOI (+/-SEM) induced by PAF at 10(-7) M to mean +/- SEM values of 30.6 +/- 6.5, 39.3 +/- 6.0, 12.1 +/- 4.8, and 41.5 +/- 6.0, respectively. The simultaneous vasoconstrictor action of 10(-7) M PAF was expressed as the experimental-to-baseline ratio, with the baseline diameter adjusted to a value of 1. PAF constricted the arterioles to a mean +/- SEM ratio of 0.30 +/- 0.07. Pretreatment with the PLC inhibitors NCDC at 10(-4) and 10(-5) M NCDC and with U-73122 at 10(-5) and 10(-6) M attenuated 10(-7) M PAF-induced vasoconstriction to mean +/- SEM diameter ratios of 0.55 +/- 0.05, 0. 48 +/- 0.06, 0.55 +/- 0.08, and 0.58 +/- 0.06, respectively. Our results demonstrate that PLC is an element of the biochemical pathway involved in PAF modulation of microvascular permeability and in PAF modulation of arteriolar diameter.

Administration, Topical↗

PubMed2000

Stimulation of NO production and of eNOS phosphorylation in the microcirculation in vivo.

The role of nitric oxide (NO) in microvascular permeability is controversial, in part because the regulation of its endothelial constitutive synthase, eNOS, has been studied in vitro but not in vivo. Our study was designed to detect the morphologic and functional presence of eNOS and to test whether eNOS could be phosphorylated by platelet-activating factor (PAF), an agent that induces hyperpermeability. Immunocytochemistry was applied using human anti-eNOS antibodies in the hamster cheek pouch (hcp). The hcp microvessels demonstrated positive reaction products in the endothelium. The functional presence of eNOS in hcp was investigated by topical application of 10(-7) M PAF to the hcp and by measuring NO production by chemiluminescence. The mean baseline value of NO release was 63.3 +/- 6.9 pmol/ml (mean +/- SE). Application of PAF led to an increase in mean NO release to 120.8 +/- 31.2 pmol/ml (P < 0.05). In another series of experiments, 10(-7) M PAF was applied topically to hcp preincubated with [(32)P]orthophosphoric acid. Immunoprecipitation and Western blots detected (32)P-labeled bands that migrated with the mobility of positive eNOS indicating phosphorylated eNOS protein. The intensity of the radioactive bands was evaluated by computer-assisted image analysis. Comparison of the net band intensities yielded a mean PAF-treated/control ratio of 1.6 +/- 0.1. Our data demonstrate the morphologic and functional presence of eNOS in the microcirculation. The data also provide evidence that the function of microvascular eNOS is subject to regulation by phosphorylation.

Animals↗

PubMed2000

Vein interposition cuffs decrease the intimal hyperplastic response of polytetrafluoroethylene bypass grafts.

PURPOSE: The modification of the distal anastomosis of polytetrafluoroethylene (PTFE) bypass grafts with vein interposition cuffs (VCs) has been reported to increase graft patency. However, the mechanisms that are responsible for this improved patency are unclear. Because intimal hyperplasia (IH) is a primary cause of prosthetic graft failure, we hypothesized that VCs affect the distal anastomosis by decreasing the IH response of the outflow artery. METHODS: Twenty-three female domestic Yorkshire pigs (mean weight, 35 kg) underwent 42 femoral PTFE bypass grafting procedures. The PTFE bypass grafts were separated into the following three groups according to distal anastomotic configuration: end-to-side anastomoses (ES), VCs, and cuffs constructed with PTFE (PCs). Four femoral arteries from two pigs served as healthy controls. At sacrifice, the grafts were perfusion fixed, and the distal anastomoses harvested at 1 and 4 weeks. The specimens were hemisected and serially sectioned to identify the heel, toe, and mid-anastomotic regions. The sections were cut into 5-microm segments and analyzed for intima and media thickness and area, intima/media area ratio, and the distribution of IH in the vein cuff. The roles of transforming growth factor-beta1 and platelet-derived growth factor-BB in IH development were assessed with immunohistochemistry. RESULTS: IH development was significantly lower at all areas of the anastomosis, with VCs compared with ES and PCs at 4 weeks (P </=.001). IH decreased in VCs from 1 to 4 weeks in all areas of the anastomosis (P </=.001). PCs showed pronounced IH at the mid-anastomosis as compared with VCs and ES (P </=.001). IH was most pronounced at the toe with ES and PCs (P </=.001). Qualitatively, VCs altered the site of IH development, sparing the recipient artery with preferential thickening of the vein cuff and formation of a pseudointima at the vein-PTFE interface. Immunohistochemistry results showed positive staining for transforming growth factor-beta1, platelet-derived growth factor-BB, and smooth muscle alpha-actin in the hyperplastic intima. CONCLUSION: PTFE bypass grafts with VCs had less IH develop than did grafts with ES and PC anastomoses. IH regression in VCs at 4 weeks suggests compensatory vessel wall remodeling mediated by the presence of the VC. Furthermore, VCs caused a redistribution of hyperplasia to the vein-PTFE interface, delaying IH-induced outflow obstruction in the recipient artery. The marked increase in IH with PCs, despite a similar geometric configuration to VCs, suggests that the biologic properties of autogenous tissue dissipate IH development. Similarly, the flow patterns in PCs and VCs should be identical, which suggests a less important role of hemodynamic forces in VC-mediated protection.

Actins↗

PubMed2000

Adenoviral-mediated expression of antisense RNA to basic fibroblast growth factor reduces tangential stress in arterialized vein grafts.

PURPOSE: The purpose of this study was to test whether basic fibroblast growth factor (bFGF) participates in arterialized vein graft remodeling. METHODS: Rabbits underwent in vivo gene transfer and carotid interposition vein grafting. Segments of external jugular vein were infected with an adenovirus that expressed antisense bFGF RNA (Ad.ASbFGF) at 1 x 10(10) PFU/mL to inhibit new synthesis of bFGF by cells in the vein graft wall. Control rabbits were treated with either adenovirus that encoded beta-galactosidase (Ad.lacZ) at 1 x 10(10) PFU/mL or vehicle (phosphate-buffered saline solution [PBS]). At 3 days, 3 grafts per treatment group were harvested for the determination of gene expression of ASbFGF RNA by reverse transcriptase-polymerase chain reaction. Rabbits were killed, and perfusion was fixed 2 months after the grafting. Total wall thickness and lumen circumference of vein grafts and normal arteries were measured in cross sections. Calculated mean tangential stress (+/-SD) for the ASbFGF-treated group and controls was compared for significance. Grafts were immunohistochemically stained to assess bFGF protein production. RESULTS: Only the grafts infected with the Ad.ASbFGF gene expressed ASbFGF RNA. Grafts that were treated with Ad.ASbFGF displayed lower tangential stress (10.9 +/- 2.3 dynes/cm(2)) than PBS alone (22 +/- 2.8 dynes/cm(2)) or Ad. lacZ-treated controls (20.6 +/- 5.4 dynes/cm(2); P <.001). Tangential stress in the Ad.ASbFGF group was comparable to a normal carotid artery (13.9 +/- 2.1 dynes/cm(2)). The difference in mean total wall thickness was significant among the 3 treatment groups: Ad.ASbFGF, 164 +/- 3.4 microm); Ad.lacZ, 100 +/- 3.3 microm; and PBS, 96 +/- 3.6 microm; P <.01). Luminal circumference was not different among the groups. The Ad.ASbFGF-treated vein graft wall was composed of thick layers of concentric smooth muscle cells and elastin fibers in contrast to the sponge-like appearance observed in control arterialized vein grafts. Reduction in bFGF protein was noted only in the Ad.ASbFGF-treated group. CONCLUSION: Inhibition of bFGF synthesis in vivo with the use of adenoviral gene transfer of antisense RNA to bFGF promotes a vein graft with decreased tangential stress while maintaining the luminal area. The vein graft wall is remodeled and qualitatively resembles an artery so that wall tangential stress in Ad.ASbFGF and normal artery are not significantly different. The lack of significant difference in lumen circumference among groups suggests that wall thickening in the Ad. ASbFGF grafts is not at the expense of luminal narrowing. Our results suggest that ASbFGF RNA expression may represent an effective strategy in limiting the failure of arterialized venous conduits.

Adenoviridae↗

PubMed1999

Dermal tissue fibrosis in patients with chronic venous insufficiency is associated with increased transforming growth factor-beta1 gene expression and protein production.

PURPOSE: Pathologic dermal degeneration in patients with chronic venous insufficiency (CVI) is characterized by aberrant tissue remodeling that results in stasis dermatitis, tissue fibrosis, and ulcer formation. The cytochemical processes that regulate these events are unclear. Because transforming growth factor-beta(1) (TGF-beta(1)) is a known fibrogenic cytokine, we hypothesized that the increased production of TGF-beta(1) would be associated with CVI disease progression. METHODS: Seventy-eight punch biopsy specimens of the lower calf (LC) and the lower thigh (LT) of 52 patients were snap frozen in liquid nitrogen and stratified into four groups according to the Society for Vascular Surgery/International Society for Cardiovascular Surgery CEAP classification (C, clinical; E, etiologic; A, anatomic distribution; and P, pathophysiology). One set of LC biopsy specimens were analyzed for TGF-beta(1) gene expression with quantitative reverse transcriptase-polymerase chain reaction: healthy skin, n = 6; class 4, n = 6; class 5, n = 5; and class 6, n = 7. A second set of biopsy specimens from the LC and LT were analyzed for the amount of bioactive TGF-beta(1) with a certified cell line 64 mink lung epithelial bioassay: healthy skin, n = 8; class 4, n = 23; class 5, n = 13; and class 6, n = 10. The location of TGF-beta(1) was determined at the light and electron microscopy level with immunocytochemistry and immunogold (IMG) labeling. Multiple comparisons were analyzed with a one-way analysis of variance and the Student-Newman-Keuls post hoc tests. The LC and LT comparisons were analyzed with a two-tailed unpaired t test. RESULTS: The TGF-beta(1) gene transcripts for control subjects and patients in classes 4, 5, and 6 were 7.02 +/- 7.33, 43.33 +/- 9.0, 16.13 +/- 7.67, and 7.22 +/- 0.56 x 10(-14) mol/microg total RNA, respectively. The transcripts were significantly elevated in class 4 patients only (P </=.05). The amount of active TGF-beta(1) in picograms/gram of tissue from LC and LT biopsy specimens as compared with healthy skin biopsy specimens were as follows: healthy skin, <1. 0 pc/g; class 4: LC, 5061 +/- 1827 pc/g; LT, 317.3 +/- 277 pc/g; class 5: LC, 8327 +/- 3690 pc/g; LT, 193 +/- 164 pc/g; and class 6: LC, 5392 +/- 1800 pc/g; LT, 117 +/- 61 pc/g. Differences between healthy skin and the skin of the patients in classes 4 and 6 were significant (P </=.05 and P </=.01, respectively). Differences between the LC and LT biopsy specimens within each CVI group were also significant: class 4, P </=.003; class 5, P </=.008; and class 6, P </=.02. Immunocytochemistry results of healthy skin showed TGF-beta(1) staining of epidermal basal cells only. CVI dermal biopsy results demonstrated positive staining in epidermal basal cells, fibroblasts, and leukocytes. Many leukocytes had positive staining of intracellular granules, which appeared morphologically similar to mast cells. IMG labeling results demonstrated gold particles in the leukocytes and collagen fibrils of the extracellular matrix. CONCLUSION: Our study indicated that activated leukocytes traverse perivascular cuffs and release active TGF-beta(1). Positive TGF-beta(1) staining results of dermal fibroblasts were observed and suggest that fibroblasts are the targets of activated interstitial leukocytes. Increased protein production, despite normal levels of gene transcripts in patients in classes 5 and 6, suggests that alternate mechanisms other than gene transcription regulate protein production. A potential mechanism for quick access and release is storage of TGF-beta(1) in the extracellular matrix. IMG labeling to collagen fibrils support this possibility. Furthermore, TGF-beta(1) was exclusively elevated in areas of clinically active disease, indicating a regionalized response to injury. These data suggest that alterations in tissue remodeling occur in patients with CVI and that dermal tissue fibrosis in CVI is regulated by TGF-beta(1).

Animals↗

PubMed1999

Hemodialysis access: influence of the human immunodeficiency virus on patency and infection rates.

PURPOSE: The complication rate for patients who are dialysis dependent and infected with the human immunodeficiency virus (HIV) and the role of viral indicators (CD4 counts) as predictors of these complications are poorly characterized. To determine the influence of HIV status and viral activity on graft patency and infection rates, we retrospectively reviewed our results. METHODS: Between June 1993 and March 1997, the charts of 104 patients (HIV+, n = 42; HIV-, n = 62) who required 112 hemodialysis access grafts were reviewed. Of the 112 procedures, 55 (48%) were autologous arteriovenous fistulae (AVF) procedures (HIV+, n = 23; HIV-, n = 32) and 57 (52%) were prosthetic expanded polytetrafluoroethylene grafting procedures (HIV+, n = 27; HIV-, n = 30). Transcutaneous catheter procedures were excluded from the study. The autologous AVF procedures consisted of direct and transposed AVFs. Patency rates were determined by means of life-table analysis. Infection rates and CD4 counts were compared with the chi2 test and the Fisher exact test. Significance was accepted at a P value of.05 or less. RESULTS: The cumulative 12-month and 24-month patency rates for prosthetic grafts in patients who were HIV+ were 49% and 21%, respectively, versus 77% and 45% for patients who were HIV-. The differences in the prosthetic graft patency rates between these two groups were significant (P </=.05). The cumulative 12-month and 24-month patency rates for autologous AVF procedures did not differ significantly. The AVF procedure patency rates were 72% and 51%, respectively, in patients who were HIV+ versus 54% and 50% for patients who were HIV-. The prosthetic graft infection rate for patients who were HIV+ and HIV- were 30% and 7%, respectively ( P =.04). However, the infection rates in autologous AVF procedures did not differ between the groups (9% vs 0%; P>.05). The mean CD4+ cell counts were 174: CD4+ counts that were less than 200 did not correlate with or predict the development of infection (P >.05). CONCLUSION: Our data showed that prosthetic graft infection rates were increased and patency rates were decreased in patients who were HIV+ as compared with patients who were HIV- and HIV+ with autologous AVFs. There were no differences in patency rates or infection rates in patients who had undergone autologous access procedures. Long-term graft patency rates were not affected by HIV status, and CD4+ lymphocyte counts were not predictive of infection development. Because the prosthetic graft infection rates exceeded those rates of autologous access procedures, we recommend the vigorous use of autologous AVFs in all patients who are HIV+, regardless of CD4+ count.

Blood Vessel Prosthesis Implantation↗

PubMed1998

Retinoblastoma protein: a molecular regulator of chronic venous insufficiency.

PURPOSE: Chronic venous insufficiency (CVI) and varicose vein (VV) formation is characterized histologically by the transformation of smooth muscle cells (SMC) from a contractile to a secretory phenotype and by intense collagen deposition. The subcellular regulation point for these processes may be the retinoblastoma protein (pRb), a known inhibitor of cellular proliferation and regulator of differentiation. We hypothesize that pRb phosphorylation is associated with VV formation and functions as a possible subcellular regulator. METHODS: Patients were separated into two groups. Group 1 (n = 6) consisted of vein specimens obtained from patients undergoing coronary artery bypass grafting. Group 2 (n = 6) consisted of patients with symptomatic CVI and duplex confirmed refluxing greater saphenous veins (GSVs) who required GSV stripping. Western blots of GSV protein extracts were performed with anti-human pRb monoclonal antibodies and the degree of nonphosphorylated and phosphorylated pRb was determined. Results were quantified using image analysis of band intensities (computer calibrated intensity units). The ultrastructural appearance of SMCs and the vein wall architecture were qualitatively analyzed with electron microscopy in both groups. RESULTS: Phosphorylated pRb from varicose GSVs exhibited intensities of 523 +/- 188 units, while phosphorylated pRb from normal GSVs demonstrated intensities of 153 +/- 41 units (P < 0.05). SMCs in varicosed GSVs were surrounded by disorganized collagen deposits and displayed a secretory phenotype with spherical vacuolated cells. SMCs from normal GSVs appeared spindle shaped with a purported contractile phenotype and a well-structured extracellular matrix. CONCLUSION: Our data demonstrate that VV formation, in patients with CVI, is associated with phosphorylated pRb and the transformation of SMCs from a contractile to a secretory ultrastructural morphology. The data suggest that SMC dedifferentiation is regulated by pRb and the disinhibition of this protein (phosphorylation) may be an significant factor in the development of lower extremity varicosities.

Blotting, Western↗

PubMed1998

Microvascular pathophysiology of skeletal muscle ischemia-reperfusion.

Ischemia-reperfusion injury is a continuing challenge to vascular surgeons. Microvascular factors and mechanisms underlying edema and compartment syndrome provide a useful end point for analysis and planning of therapy. We review the pivotal role of endothelium-leukocyte interactions and of cytokines in the genesis of postischemic damage in muscle. Clinical considerations and future directions based on research and practice are presented.

Acute Disease↗

PubMed1997

Novel neuropeptide Y receptor antagonists block vasoconstriction in the hamster cheek pouch microcirculation.

We investigated the efficacy of novel neuropeptide Y (NPY) antagonists to inhibit the microcirculatory dynamics of NPY in the hamster cheek pouch microcirculation using intravital microscopy and computer-assisted image analysis. Changes in arteriolar diameter served as an index of vasomotor alterations. Fluorescein isothiocyanate-labeled Dextran 150 served as a tracer for measurements of macromolecular transport. GW 383 and GW 1229, two novel NPY receptor antagonists, were applied topically in separate experiments. Pretreatment with 10(-5), 10(-6) and 10(-7) M GW 383 and with 10(-6) and 10(-8) M GW 1229 attenuated the vasoconstriction induced by 10(-7) M NPY in a dose-dependent manner. Furthermore, pretreatment with 10(-7) and 10(-8) M GW 1229 significantly inhibited the 10(-9) M NPY-induced vasoconstriction. At these doses, the NPY antagonists did not alter microvascular permeability. Our results demonstrate that the novel NPY antagonists inhibit the vasoconstriction induced by NPY in the hamster check pouch microcirculation. We suggest that the inhibition is due to binding of antagonists to Y1-type NPY receptors.

Animals↗

PubMed1997

Permselectivity of angiogenic microvessels following alteration of the endothelial fiber matrix by oligosaccharides.

The endothelial glycocalyx, which is composed of integral and peripheral glycoconjugates, forms a fibrous matrix that confers macromolecular sieving properties on the microvascular wall. Changes in pore size within the matrix may regulate macromolecular access to the paracellular and/or vesicular transendothelial pathways. We tested the hypothesis that modifications of the endothelial glycocalyx might play a role in the ontogeny of endothelial permselectivity in proliferating microvessels of the chick chorioallantoic membrane (CAM). Accordingly, we evaluated the effects of Dolichos biflorus agglutinin (DBA) or Arachis hypogaea agglutinin (PNA) lectin binding, and N'N'diacetylchitobiose or hydroxyethyl starch polysaccharide (HES) incorporation on CAM endothelial restriction of FITC-dextrans 40 or 150 at Days 4.5 and 5.0 of development. Extravasation of FITC-dextrans was determined by recording their perivascular interstitial intensities. Following DBA, PNA, and N'N'diacetylchitobiose administration, interstitial accumulation of the tracers near first-order pre- and postcapillaries, and surrounding the capillaries, was similar to that of controls at both Days 4.5 and 5.0. At Day 4.5, pretreatment with HES significantly decreased extravasation of FITC-dextran 40. Thus, retention of HES molecules within the glycocalyx might tighten the matrix, and reduce access of dextran 40 to transendothelial pathways across the angiogenic microvessels.

Animals↗

PubMed1997

Morphometric assessment of the dermal microcirculation in patients with chronic venous insufficiency.

PURPOSE: Ultrastructural assessments of the dermal microcirculation in patients with chronic venous insufficiency have been limited to qualitative morphologic descriptions of venous ulcer edges or venous stasis dermatitis. The purpose of this investigation was to quantify differences in endothelial cell structure and local cell type with emphasis on leukocytes and their relationship to arterioles, capillaries, and postcapillary venules (PCVs). METHODS: Two 4.0 mm punch biopsies were obtained from areas of dermal stasis skin changes in the gaiter region of the leg, as well as from noninvolved areas of skin in the ipsilateral thigh, from 35 patients: CEAP class 4 (11 patients), class 5 (9 patients), class 6 (10 patients), and five normal skin biopsies from patients without chronic venous insufficiency. Electron microscopy was performed on sections at 6700x and 23,800x magnification. At 6700x endothelial cell thickness was determined, and the number of fibroblasts, leukocytes, and mast cells were recorded relative to their proximity to arterioles, capillaries, and PCVs. Similarly, at 23,800x endothelial cell vesicle density, interendothelial junctional widths, and basal lamina thickness (cuff width) were measured. Preliminary evaluation for the presence of transforming growth factor-beta 1 (TGF-beta 1) was performed on three patients using reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Quantitative measurements demonstrated increased mast cell content for class 4 and 5 patients around arterioles and PCVs and increased macrophage numbers for class 6 patients around PCVs (p < 0.05). Fibroblasts were the most common cells observed; however, no differences were demonstrated between groups. No differences were observed in interendothelial junctional widths or vesicle densities in arterioles, capillaries, or PCVs. Basal lamina thickness was increased only at the capillary level (p < 0.05). The results of RT-PCR for TGF-beta 1 messenger RNA were positive in the three patients studied. CONCLUSIONS: Our data suggest that (1) mast cells play a role in the pathogenesis of chronic venous insufficiency; (2) the effects of mast cells, macrophages, or both may be mediated in part by TGF-beta 1; and (3) capillary cuff formation is not associated with widened interendothelial gap junctions, but may be a result of enhanced vesicular transport rate or conformational changes in the interendothelial glycocalyx.

Aged↗

PubMed1996

Time course of leukocyte adhesion to endothelium in ischemia-reperfusion.

Adhesion of leukocytes (L) to microvascular endothelium (E) is a required step in the L-E interaction leading to tissue injury in ischemia-reperfusion. To assess the optimum period for therapy aimed at ameliorating negative effects of this required step, we investigated the time course of L-E adhesion in the hamster cheek pouch using 2 hr of ischemia and 1 hr of reperfusion in our model of I-R injury (Am. J. Physiol-261: 1626, 1991). Leukocytes adhering (stationary for > or = 30 sec) to postcapillary venules (15-30 microns in diameter) were counted after labeling with acridine orange. Prior to the induction of ischemia, there were no significant differences in the number of adherent leukocytes in each area chosen for study (1.9 +/- 0.6 vs 2.0 +/- 0.3; mean number of leukocytes/100-microns vessel length +/- SD). After 10 and 20 min of reperfusion there was no significant difference in leukocyte adhesion in the ischemic area relative to the control (2.7 +/- 0.5 vs 2.8 +/- 0.8, and 5.3 +/- 2.8 vs 2.4 +/- 0.6, respectively). Leukocyte adherence increased significantly after 30 min of reperfusion and remained elevated at 1 hr of reperfusion in the postischemic area relative to the nonischemic control area (7.8 +/- 1.3 vs 3.6 +/- 0.6, and 8.3 +/- 0.8 vs 4.1 +/- 0.6, respectively; P < 0.01). Leukocyte adhesion in the postischemic area after 30 min reperfusion was not significantly different from the adhesion at the end of 1 hr reperfusion. These data suggest that (1) peak leukocyte adhesion occurs after 30 min of normal reperfusion and (2) postischemic therapeutic intervention may be most beneficial when instituted within this early time period.

Animals↗

PubMed1996

Platelet-activating factor modulates leukocyte adhesion to endothelium in ischemia-reperfusion.

Platelet-activating factor (PAF) is an important proinflammatory phospholipid that may be involved in modulating leukocyte-endothelium (L-E) adhesion in ischemia-reperfusion (I-R). We investigated the role of PAF receptors in postischemic reperfusion using our model of I-R in the hamster cheek pouch microcirculation. The model allows for measurements of ischemic and nonischemic areas in the same preparation. We assessed the increase in the number of adhering leukocytes (per 100-microns vessel length) as an index of I-R-induced microvascular dysfunction. To test the influence of endogenous PAF, we administered WEB 2086 (2 mg/kg iv), a PAF receptor inhibitor. In the control I-R group (Group 1), the number of adhering leukocytes (mean +/- SEM) increased from 3.3 +/- 0.7 at baseline to 9.5 +/- 1.0 at the end of 1 hr of reperfusion. In a second group of animals (Group 2) receiving WEB 2086 prior to ischemia, there was no significant difference between the preischemic baseline and the values recorded after 1 hr of reperfusion (3.5 +/- 0.5 vs 3.8 +/- 0.5). In a third group of hamsters (Group 3) receiving WEB 2086 10 min prior to reperfusion, there was no significant difference between the baseline values and those at 1 hr of reperfusion (2.3 +/- 0.3 vs 2.3 +/- 0.2). Similarly, in Groups 2 and 3, with WEB 2086 treatment, there was no significant difference between the values measured in the ischemic areas and their time-matched nonischemic areas at the end of 1 hr of reperfusion (Group 2, 3.8 +/- 0.5 vs 3.0 +/- 0.4; Group 3, 2.3 +/- 0.2 vs 3.2 +/- 0.4). Our data demonstrate (1) an important role for PAF receptors in modulating L-E adhesion in I-R and (2) that blockade of PAF receptors either prior to ischemia or prior to reperfusion reduces adhesion of leukocytes to endothelium. Our results suggest that PAF receptor blockade in postischemic tissues could be a new therapeutic approach to decrease the impact of ischemia-reperfusion injury.

Animals↗

PubMed1996

Platelet-activating factor and nitric oxide mediate microvascular permeability in ischemia-reperfusion injury.

Increased microvascular permeability is a hallmark of ischemia-reperfusion (I/R) injury. We hypothesized that platelet-activating factor (PAF) and nitric oxide (NO) are involved in the extrvasation of macromolecules in I/R injury. To block endogenous PAF, we used a PAF-receptor antagonist (WEB 2086; 2 mg/kg, i.v). To inhibit endogenous nitric oxide, we employed L-NG-monomethyl arginine (10(-5) M L-NMMA), a NO synthase inhibitor. We assessed microvascular permeability to FITC-dextran 150 by measuring changes in integrated optical intensity (delta IOI) using computer-assisted image analysis in the hamster cheek pouch. We examined one area of ischemia and one control area in each pouch. Ischemia was induced for 2 hr and was followed by 1 hr of reperfusion. Six groups were investigated. Group 1 (n = 5) had no pharmacologic intervention; Group 2 (n = 5) received WEB 2086 15 min before reperfusion; Group 3 (n = 5) received WEB 2086 at reperfusion; Group 4 (n = 5), WEB 2086 was infused 15 min after the onset of reperfusion. Group 5 (n = 3) received topical L-NMMA (30 min prior to reperfusion and continuously for the remainder of the experiment). Group 6 (n = 3) received both L-NMMA (as in Group 5) and WEB 2086 (administered 15 min after reperfusion). In Group 1, I/R increased the mean (+/- SEM) delta IOI value from 61.5 +/- 11.1 to 127.2 +/- 26.1. WEB 2086 inhibited the increase in delta IOI at each time point. Similarly, the groups given L-NMMA alone and L-NMMA + WEB 2086 showed no difference between ischemic and control groups. Our data demonstrate that (1) PAF and nitric oxide are involved in the permeability changes associated with the microvascular dysfunction of ischemia-reperfusion injury; (2) inhibitors of PAF and nitric oxide synthase are effective in attenuating macromolecular extravasation when given during ischemia or after initiation of reperfusion.

Animals↗

PubMed1996

Inhibition of white blood cell adhesion at reperfusion decreases tissue damage in postischemic striated muscle.

PURPOSE: To determine the impact of white blood cell (WBC)-endothelium adhesion on tissue damage in the setting of ischemia-reperfusion injury in striated muscle. METHODS: The cremaster muscle of four groups of anesthetized Sprague-Dawley rats was subjected to 4 hours of global, warm (37 degrees C) ischemia and 2 hours of reperfusion. At reperfusion two groups of animals received intravenous injections of monoclonal antibodies directed against either CD11b/CD18 (1B6) or ICAM-1 (1A29). The remaining two groups of animals received saline injections (NoRx) or nonreactive IgG1. In vivo light microscopic techniques were used to determine WBC adherence (number of WBCs per 100 microns postcapillary venules) at different intervals of reperfusion. Muscle viability was assessed with computer-assisted image analysis by measuring the optical intensity of transilluminated muscles after incubation with nitroblue tetrazolium. RESULTS: Our results (mean +/- SEM) demonstrate a significant increase in the number of adherent WBCs relative to baseline (8.0 +/- 0.5) after 4 hours of global ischemia in animals receiving NoRx or IgG1. The significant increase occurred at 30 minutes of reperfusion (17.6 +/- 0.6 and 17.4 +/- 0.4 for NoRx or IgG1, respectively) and was sustained for the duration of the experiment. This increase in adherence was attenuated by 1B6 and 1A29 (12.2 +/- 2.2 and 12.4 +/- 0.8, respectively; p < 0.05 compared with NoRx and IgG1). The decrease in WBC adhesion was associated with a decrease in reperfusion injury to the muscle, as indicated by lower optical intensity values for the 1B6 and 1A29 groups (123 +/- 3 and 129 +/- 2) compared with the NoRx and IgG1 groups (151 +/- 2 and 158 +/- 4). CONCLUSIONS: Our data support an important role for WBCs in the pathogenesis of ischemia-reperfusion injury. Interfering with the WBC-endothelium interactions by using monoclonal antibodies directed against WBCs and endothelial cell adhesion molecules may help to limit ischemia-reperfusion injury.

Animals↗

PubMed1996

Protein kinase C modulates microvascular permeability through nitric oxide synthase.

Protein kinase C (PKC) serves important functions in signal transduction. We hypothesized that PKC modulation of microvascular permeability to macromolecules is mediated by nitric oxide (NO). To test this hypothesis, we stimulated PKC topically with 10(-7) M phorbol 12,13-dibutyrate (PDBu) in the hamster check pouch microcirculation. NG-monomethyl-L-arginine (L-NMMA) at 10(-4) M was superfused in a bicarbonate buffer solution throughout the experiment to inhibit the activity of NO synthase. We evaluated changes in transport of fluorescein isothiocyanate-labeled 150,000 mol wt dextran by integrated optical intensity (IOI) using intravital fluorometry and computer-assisted digital image analysis. Postcapillary areas were recorded. PDBu increased IOI from baseline to a value of 46.8 +/- 6.3 units (+/- SE). Pretreatment with L-NMMA decreased the PDBu-stimulated increment to 10.8 +/- 0.9 units. These results demonstrate that PKC-activated modulation of macromolecular transport operates through a mechanism involving the production of NO.

Animals↗