Walter N. Durán

The significance of calf muscle pump function in venous ulceration.

PURPOSE Patients with clinically evident chronic venous insufficiency were evaluated to relate the degree of insufficiency and calf muscle pump dysfunction to venous ulceration. METHODS Sixty-nine limbs in 55 patients with chronic venous insufficiency by Society for Vascular Surgery/International Society for Cardiovascular Surgery Classification were compared in three groups: classes 1 and 2 with no history of ulceration (19 limbs); class 3 with healed ulceration (20 limbs); and class 3 with active ulcers (30 limbs). Air plethysmography measurements of outflow fraction, venous volume, venous filling time, venous filling index, ejection fraction, ejection volume, residual volume fraction, and residual volume were made. In 62 of the 69 limbs, color-flow duplex ultrasonography was used to determine the pattern of reflux. RESULTS The outflow fraction was normal in 84%, 75%, and 77% of nonulcerated, healed, and ulcerated limbs. The venous filling index was abnormal in most limbs (nonulcerated 95%, healed 90%, ulcerated 98%) but not significantly different among groups. Differences in calf muscle pump function were significant. Ulcerated limbs had significantly poorer ejection fractions (p = 0.0002) and greater residual volume fractions (p = 0.0006) than nonulcerated or healed limbs. By ultrasonography, deep and superficial vein incompetence was present in most limbs and was not statistically different among groups. Although venous insufficiency was not measurably different among groups, limbs with active venous ulcers had significantly poorer calf muscle pump function than those with healed ulcers or with no history of ulceration. CONCLUSION Venous insufficiency is necessary but not sufficient to cause ulceration, and a deficiency of the calf muscle pump is significant to the severity of venous ulceration.

Microvascular transport and endothelial cell alterations preceding skeletal muscle damage in ischemia and reperfusion injury

We determined the leakage of macromolecules using FITC-dextran-150 as a tracer and measured the extent of no-reflow phenomenon by video field analysis. The cremaster muscle of anesthetized rats was fashioned as a single layer, splayed on a lucite chamber and suffused with bicarbonate solution at 35 degrees C. After a 1 hour period of baseline data collection, ischemia was produced by cross-clamping the cremasteric vascular pedicle for periods of 30 minutes and 2 hours in separate experiments. Macromolecular leakage was visualized after reinstitution of perfusion. Leakage occurred at postcapillary venules 15 to 50 micron in diameter and quickly spread to the interstitium. The magnitude of leakage decreased as a function of time with continuous buffer suffusion, but remained higher than in the control period. No reflow occurred in approximately 30 percent of the muscle microvasculature upon reperfusion. The no-reflow values at 30 minute and 2 hour periods of ischemia were significantly different from the control values but were not from each other. Electron micrographs demonstrated endothelial cell swelling and migration of leukocytes and normal myocytes after 1 hour of reperfusion following 2 hours of ischemia. Our results demonstrate that permeability changes, occurrence of no reflow, and leukocyte migration precede the onset of damage to skeletal muscle in ischemia and reperfusion injury.

The NO cascade, eNOS location, and microvascular permeability

The nitric oxide (NO) cascade and endothelial NO synthase (eNOS) are best known for their role in endothelium-mediated relaxation of vascular smooth muscle. Activation of eNOS by certain inflammatory stimuli and enhanced NO release have also been shown to promote increased microvascular permeability. However, it is not entirely clear why activation of eNOS by certain vasodilatory agents, like acetylcholine, does not affect microvascular permeability, whereas activation of eNOS by other inflammatory agents that increase permeability, like platelet-activating factor, does not cause vasodilation. In this review, we discuss the evidence demonstrating the role of eNOS in the elevation of microvascular permeability. We also examine the relative importance of eNOS phosphorylation and localization in its function to promote elevated microvascular permeability as well as emerging topics with regard to eNOS and microvascular permeability regulation.

Nitroglycerin-Induced S-nitrosylation and Desensitization of Soluble Guanylyl Cyclase Contribute to Nitrate Tolerance

Nitrates such as nitroglycerin (GTN) and nitric oxide donors such as S-nitrosothiols are clinically vasoactive through stimulation of soluble guanylyl cyclase (sGC), which produces the second messenger cGMP. Development of nitrate tolerance, after exposure to GTN for several hours, is a major drawback to a widely used cardiovascular therapy. We recently showed that exposure to nitric oxide and to S-nitrosothiols causes S-nitrosylation of sGC, which directly desensitizes sGC to stimulation by nitric oxide. We tested the hypothesis that desensitization of sGC by S-nitrosylation is a mechanism of nitrate tolerance. Our results established that vascular tolerance to nitrates can be recapitulated in vivo by S-nitrosylation through exposure to cell membrane-permeable S-nitrosothiols and that sGC is S-nitrosylated and desensitized in the tolerant, treated tissues. We next determined that (1) GTN treatment of primary aortic smooth muscle cells induces S-nitrosylation of sGC and its desensitization as a function of GTN concentration; (2) S-nitrosylation and desensitization are prevented by treatment with N-acetyl-cysteine, a precursor of glutathione, used clinically to prevent development of nitrate tolerance; and (3) S-nitrosylation and desensitization are reversed by cessation of GTN treatment. Finally, we demonstrated that in vivo development of nitrate tolerance and crosstolerance by 3-day chronic GTN treatment correlates with S-nitrosylation and desensitization of sGC in tolerant tissues. These results suggest that in vivo nitrate tolerance is mediated, in part, by desensitization of sGC through GTN-dependent S-nitrosylation.

Endothelial nitric oxide synthase regulates microvascular hyperpermeability in vivo

Nitric oxide (NO) is an important regulator of blood flow, but its role in permeability is still challenged. We tested in vivo the hypotheses that: (a) endothelial nitric oxide synthase (eNOS) is not essential for regulation of baseline permeability; (b) eNOS is essential for hyperpermeability responses in inflammation; and (c) molecular inhibition of eNOS with caveolin‐1 scaffolding domain (AP‐Cav) reduces eNOS‐regulated hyperpermeability. We used eNOS‐deficient (eNOS−/−) mice and their wild‐type control as experimental animals, platelet‐activating factor (PAF) at 10−7m as the test pro‐inflammatory agent, and integrated optical intensity (IOI) as an index of microvascular permeability. PAF increased permeability in wild‐type cremaster muscle from a baseline of 2.4 ± 2.2 to a peak net value of 84.4 ± 2.7 units, while the corresponding values in cremaster muscle of eNOS−/− mice were 1.0 ± 0.3 and 15.6 ± 7.7 units (P < 0.05). Similarly, PAF increased IOI in the mesentery of wild‐type mice but much less in the mesentery of eNOS−/− mice. PAF increased IOI to comparable values in the mesenteries of wild‐type mice and those lacking the gene for inducible NOS (iNOS). Administration of AP‐Cav blocked the microvascular hyperpermeability responses to 10−7m PAF. We conclude that: (1) baseline permeability does not depend on eNOS; (2) eNOS and NO are integral elements of the signalling pathway for the hyperpermeability response to PAF; (3) iNOS does not affect either baseline permeability or hyperpermeability responses to PAF; and (4) caveolin‐1 inhibits eNOS regulation of microvascular permeability in vivo. Our results establish eNOS as an important regulator of microvascular permeability in inflammation.

Dermal tissue fibrosis in patients with chronic venous insufficiency is associated with increased transforming growth factor-β1 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).

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.

Refinements in the ultrasonic detection of popliteal vein reflux

Color-flow and duplex ultrasonography were used to determine the optimal method for documenting venous valvular reflux. Popliteal veins were examined in 10 normal limbs and 11 limbs with clinical evidence of chronic venous insufficiency (CVI). Peak reflux velocity (spectral) and duration of reflux (spectral and color) were measured with the patient in supine and standing positions, with manual and pneumatic compression applied sequentially to thigh and calf. Manual and pneumatic compression produced equivalent reflux velocity and duration. In normal limbs peak reflux velocity was always less than 22 cm/sec, with a mean reverse flow duration of 0.3 sec +/- 0.03 (SEM). In limbs with CVI, reflux velocity varied widely among protocols. Reflux duration and velocity were greater in the supine position than in the standing position for both normal limbs and limbs with CVI (p < 0.04). Duration was significantly increased for thigh versus calf compression in normal limbs (p < 0.001) but decreased in limbs with CVI (p < 0.003). Methods that used thigh compression or supine position were less capable of discriminating normal limbs from limbs with CVI. Standing calf compression provided the greatest rates of sensitivity (91%), specificity (100%), and accuracy (95%). Compared with spectral Doppler scanning, color-flow ultrasonography produced a consistently shorter reflux duration (p < 0.001). In limbs with CVI with a mean spectral duration of 2.5 sec +/- 0.2 (SEM), mean color Doppler duration was 0.7 sec shorter. Our results demonstrate that popliteal vein incompetence is identified optimally by reflux duration after standing calf compression; adequate manual compression is sufficient to identify reflux; color-flow Doppler ultrasonography may underestimate reflux duration.

Acupuncture Reduces Experimental Renovascular Hypertension Through Mechanisms Involving Nitric Oxide Synthases

Objective: To test the hypothesis that acupuncture on stomach 36 point (ST‐36) reduces hypertension by activating nitric oxide synthase signaling mechanisms.

Dose-related effects of adenosine and bradykinin on microvascular permselectivity to macromolecules in the hamster cheek pouch.

The hamster cheek pouch preparation was used to assess microvascular permselectivity responses to three vasodilating agents: bradykinin, adenosine, and papaverine. Fluorescein isothiocyanate-dextran 150 was injected intravenously as a macromolecular tracer. To quantify changes in permeability, we calculated fluorochrome clearance values from the ratio of suffusate to plasma fluorescein isothiocyanate-dextran 150 concentration. The microcirculation was recorded on videotape, using epifluorescence and bright-field light microscopy. Topical application of bradykinin elicited dose-dependent increases in macromolecular permeability. Adenosine also augmented permeability in a dose-dependent fashion. The increases in tracer clearance, relative to control, were 9.4 nl/min for 10−5 M adenosine and 39.4 nl/min for 10−4 M adenosine. The standard error for these doses was 1.5 nl/min. Adenosine, 10−6 M, did not alter permeability. The increment in clearance induced by 10−4 M was comparable to that of bradykinin, 8 × 10−7 M. Pretreatment with phenidone had no effect on the permeability response mediated by 10−5 M adenosine. Topical application of papaverine enhanced the transvascular exchange of macromolecules in one-half of the preparations examined. Comparable doses of adenosine were approximately three times as effective. This study indicated that adenosine, like bradykinin, is capable of modifying microvascular permeability responses in the hamster cheek pouch. This modulatory effect appears to be due to a direct action on the postcapillary microvascular membrane.