Experimental Venous Hypertension Research Articles

A pivotal role of the vascular endothelial growth factor signaling pathway in the formation of venous hypertension-induced dural arteriovenous fistulas

Dural arteriovenous fistulas (DAVFs) are associated with venous hypertension. Numerous studies have revealed high expression levels of vascular endothelial growth factor (VEGF) in human DAVF specimens, as well as in animal models of experimental venous hypertension. The objective of the present study was to clarify whether the VEGF signaling pathway is important in the development of DAVFs. Rats (n=216) were randomly divided into six groups. In the rats from five groups (groups A and C-E, n=45 in each group; group B, n=12), experimental venous hypertension was induced by right common carotid artery (CCA)-external jugular vein (EJV) anastomosis, superior sinus occlusion and left transver sinus occlusion, while the remaining group (group F, n=24) underwent sham surgery. The rats in group A received a VEGF recombinant adenovirus injection into the distal section of the right EJV 30 min prior to anastomosis of the CCA and EJV. An equivalent control adenovirus was injected into the right EJV of group B rats prior to anastomosis. The rats in group C received no virus prior to anastomosis and no medicine subsequent to surgery. The group D rats were lavaged with Vatalanib, a VEGF receptor (VEGFR) inhibitor, and the group E rats were lavaged with an equal quantity of saline weekly following surgery. Six rats from groups A-E and one rat from group F were sacrificed in the first, second, fourth and twelfth weeks after surgery for immunohistochemical analysis of VEGF expression and analysis of microvessel density. Cerebral angiography was performed on the remaining rats in each group on the twelfth week after surgery. The results revealed that following transfection with VEGF recombinant adenovirus, angiogenesis in the dura mater of venous hypertensive rats was increased subsequent to the increase in the VEGF expression levels of the brain and dura mater. The rate of DAVF induction by venous hypertension was significantly reduced by the VEGFR antagonist due to reduced angiogenesis in the dura mater. In conclusion, VEGF and its receptor may be important in the formation of venous hypertension-induced DAVFs.

Microcirculation Disturbances in Patients with Venous Ulcer before and after Healing as Assessed by Laser Doppler Flux-Metry

We studied whether a characteristic pattern of laser Doppler flux (LDF) could be identified in perimalleolar skin of patients with venous ulcer before and after ulcer healing. Nine subjects with venous ulcer that healed after conservative treatment in 1-6 months and nine healthy persons were included in the study. Microcirculation investigations of laser Doppler flux (LDF) were carried out before and after ulcer healing at rest, upon arterial occlusion, during thermal stimulation and during experimental venous hypertension. Resting LDF expressed as median and range (in arbitrary perfusion units) was significantly higher in patients with venous ulcer in comparison to healthy subjects: 60.6 (40.2-156.5) vs. 9.2 (6.5-19.5), p=0.008. During thermal stimulation and during postischemic reactive hyperemia, absolute values of LDF were slightly but significantly higher in patients with venous ulcer than in healthy subjects, but indices of hyperemic reactivity were very low in patients (median postischemic LDF increase to 101.8 vs. 450.0% in healthy controls, p=0.008; and thermally induced LDF increase to 125.5 vs. 881.5% in healthy controls, p=0.008. Experimental venous hypertension (cuff pressure 40 and 70 mmHg, respectively) led to an equally pronounced relative reduction of LDF in healthy persons as in patients with venous ulcer before and after ulcer healing, but the absolute values of flow remained about six to seven times higher in patients when compared to healthy subjects. Hyperemic reactivity and venoarteriolar response did not change after ulcer healing. Elevated basal LDF and preserved maximal LDF during reactive hyperemia were found in perimalleolar skin of patients with venous ulcers that eventually healed. The relative venoarteriolar response was preserved in spite of elevated absolute values of LDF during experimental venous hypertension. These hemodynamic characteristics remained unchanged after epithelialization of venous ulcers.

Fibrinolytic and biochemical factors in patients with venous insufficiency

Long-term metabolic changes in vein tissue of the limbs and feet have been reported in the course of chronic venous insufficiency (CVI). Low fibrinolytic potential was first demonstrated using fibrin-embedded vein rings (Todd's biopsy), then in blood via increased euglobulin lysis time. This defect is especially pronounced when blood is taken from foot veins of patients with grades 5 and 6 of CVI or skin lesions. Haemorrheological disorders such as increased blood viscosity and red cell aggregation, related mainly to high fibrinogen levels, have also been shown in patients with CVI. Such disorders, particularly observed in blood from foot veins, play an important role in the interaction between the blood cell components and the vessel wall. More recently, the margination and adhesion of white cells to the endothelium and the subsequent activation of white cells have been assumed. Adhesion molecules such as ICAM-1, VCAM-1 and P-selectin have been identified surrounding skin ulceration. There is also evidence that markers of neutrophil degranulation reach high levels in blood taken from foot veins after exposure to experimental venous hypertension. White cell activation results in elevated plasma levels of L-selectin, increased production of oxygen free radicals, pseudopods, CD11b integrin, platelet-monocyte aggregates and abnormal lymphocyte L-selectin. Increased nitric oxide, presumably due to monocyte activation, as well as increased urine haemosiderin related to skin microvessel leakage have also been reported. VCAM-1 and metalloproteinase MMP9 activity have been stated as sensitive markers in relation to endothelium activation and white cell trapping, which follows hypoxia and/or alterations in wall shear stress, mainly in skin venulae and in the region of valves.

EVALUATION OF BLOOD FLOW DISTURBANCES IN DIABETIC PATIENTS BY LASER DOPPLER FLOWMETRY

Background. The aim of this study was to investigate patients with non-insulin dependent diabetes by laser Doppler flowmetry in order to asses changes in the peripheral flow regulation during hemodynamics tests as evidence of impaired auto-regulation of the microcirculation. Methods. 2 groups of subjects were incorporated in the study. The first group included 20 patients with diabetes without electromyographical signs of neuropathy and without peripheral arterial occlusive disease while the second (control) group included 20 healthy subjects. Microcirculation investigations were carried out at rest and after hemodynamics tests. Blood flow was measured using a laser Doppler flowmetry. Changes in the flow minus the »biological zero« value were measured after 3 minutes of artificial arterial occlusion, after deep inspiration and by experimental venous hypertension (40 and 70 mm Hg) which was used to provoke venoarteriolar reflex. Results. Resting flow was significantly higher in diabetic patients compared to the healthy subjects (p < 0.01). The vasoconstrictor response to deep inspiration and experimental venous hypertension, cuff pressure 40 mm Hg, was significantly impaired in diabetic patients as compared to the healthy subjects (p < 0.001 vs. p < 0.01). The maximal hyperemia after artificial arterial occlusion was significantly reduced in diabetic patients in comparison with the healthy controls (p < 0.01). Conclusions. Laser Doppler flowmetry provides a non-invasive technique for evaluating microcirculating disturbances at a relatively stage in diabetes, long before the initial clinical presentation of peripheral arterial occlusive disease or neuropathy.

Iodoamphetamine (IMP) uptake in the brain is increased after experimental cerebral venous hypertension in the rat.

Cerebral venous hypertension (VH) is a pathological condition associated with arteriovenous malformation, and is an important determinant factor for clinical symptom and outcome. Using a rat whole brain VH model created by a carotid-jugular fistula and contralateral jugular vein ligation we measured superior sagittal sinus pressure (SSSP) and cerebral blood flow (CBF) by the 14C-iodoamphetamine (IMP) indicator fractionation technique under isoflurane anesthesia with controlled ventilation. SSSP was significantly elevated in the fistula group (15 +/- 1 mmHg) (mean +/- SEM) compared to the vessel occlusion (5 +/- 1) and sham operated groups (5 +/- 1) (P < 0.05). Mean absolute CBF in the fistula (103 +/- 3 ml/ min/100 g) and vessel occlusion (90 +/- 4) groups were elevated compared to the sham group (77 +/- 3) (P < 0.05). Physiological parameters (mean blood pressure, heart rate, and blood gas analysis) before measurement were not different. In this VH model we found increases in SSSP and IMP uptake in the brain. We assume the mechanism of IMP uptake increase in VH animal may be slow circulation with a possible change of first pass extraction of IMP.

Microcirculation disturbances in different stages of chronic venous insufficiency

Summary Objectives: The aim of this study was to investigate patients with different clinical stages of chronic venous insufficiency (CVI) by laser Doppler fluxmetry (LDF) in order to asses whether these clinical entities correspond to specific flow regulation. Methods: 3 groups of subjects were incorporated in the study. Microcirculation investigations were carried out at rest and after hemodynamics tests. Changes in the laser Doppler flux minus the biological zero value were measured after 3 minutes of arterial occlusion and by experimental venous hypertension (40 and 70 mmHg) and expressed relatively to the pretest resting value. Results: Resting LDF was significantly higher in patients with CVI II and CVI III stage as compared to the both groups with healthy subjects and the group with CVI I stage (p &lt;0.001). In patients with CVI II and CVI III stage LDF remained nearly unchanged after arterial occlusion (6% increase vs. 342% in healthy subjects and 214% increase in patients with CVI I (p &lt;0.001 vs. p &lt;0.001). Experimental venous hypertension (cuff pressure 70 mmHg) led to profound reduction of flux in all 3 groups (I: –90%; II: –91.7%; III: –91.4%). At the same time, absolute LDF values during experimental venous hypertension (40 and 70 mmHg) were significantly higher in patients with CVI II and III in comparison to patients with CVI I and healthy subjects (p &lt;0.001 vs. p &lt;0.001). Conclusion: It seems that in severe CVI patients the venoarteriolar reflex, despite being comparable in magnitude to that in healthy subjects, may nonetheless be insufficient to reduce LDF during experimental venous hypertension to the level similar to that in healthy subjects and patients with CVI I.

Microcirculatory Dysfunction in Chronic Venous Insufficiency (CVI)

The elevated ambulatory pressure in the peripheral venous system of chronic venous insufficiency (CVI) patients manifests itself not only in the form of disturbed macrocirculation but also and particularly in microangiopathic changes. For this reason, it is closely correlated with trophic disorders of the skin and can ultimately lead to ulceration. Using microcirculation research techniques, we are able to provide clear evidence of a typical microangiopathy in chronic venous insufficiency. Fifty CVI in Widmer stages I, II, and III were examined with fluorescence video microscopy, intravital video capillaroscopy, transcutaneous oxygen partial pressure measurement, TcpO2 and laser Doppler flowmetry. The effects of compression therapy with individually fitted compression stockings on capillary morphology were studied over a period of 4 weeks in 20 CVI patients in Widmer stages I and II. The capillary pressure was measured during simulated muscle contraction using a servo-null micropressure system. We periodically drew blood from the dorsalis pedis vein and a brachial vein of 11 healthy test persons and 8 patients with stage III CVI during experimental venous hypertension in order to evaluate the expression pattern of leukocyte adhesion molecules involved in inflammation: LFA-1 (CD11a), Mac-1 (CD11b), p150,95 (CD11c), CD18, VLA-4 (CD49d), and L-selectin (CD62L). In the same patients, we used immunohistochemical methods to examine clinically unaffected skin and the skin near an ulcer, focusing on the adhesion molecules ICAM-1, VCAM-1, and E-selectin. The microangiopathic changes observed with worsening clinical symptoms include a decrease in the number of capillaries, glomerulus-like changes in capillary morphology, a drop in the oxygen content (tcpO2) of the skin, increased permeability of the capillaries to low-molecular-weight substances, increased laser Doppler flux reflecting elevated subcutaneous flow, and diminished vascular reserve. These microangiopathic changes worsen in linear proportion to the clinical severity of chronic venous insufficiency. In patients with venous ulcerations, the baseline expression of LFA-1 and VLA-4 on lymphocytes, Mac-1 expression on the myeloid cell line, and L-selectin expression on all three cell lines was not significantly different form that in healthy controls. During orthostatic stress, there was a significant reduction in the expression of L-selectin in blood cells collected at foot level in the controls (p=0.002), but not in the patients. Clinical improvement by compression therapy was accompanied by an increase in the number of nutritive capillaries, while the diameter of the capillaries and the dermal papillae was reduced. When ulcers healed in a short period (<6 weeks), we observed a concomitant increase in the number of capillaries (p<0.05). Microangiopathy appears before tropic disorders of the skin develop. Even trophically normal skin areas may have dilated nutritive capillaries, an early sign of disturbed skin perfusion. These changes represent a plausible explanation for the development and to recurrency tendency of venous ulcers. The reduced expression of lymphocytic L-selectin in healthy controls during the orthostatic stress test may be an indication that the cells are activated by venous stasis. Clinically effective therapeutic measures improve the impaired microcirculation of the skin in the ankle area.

Venoarteriolar response to experimental venous hypertension in legs with chronic venous insufficiency and in healthy legs, measured using a double-wavelength laser Doppler technique.

The venoarteriolar response (VAR) of the skin in legs caused by experimental venous hypertension was measured using a new, double-wavelength laser Doppler probe technique (543 nm and 780 nm). This enables the measurement of the laser Doppler flux in the superficial and deep layers of the skin simultaneously. The recordings were obtained from the leg with the patient in a recumbent position with a sphygmomanometer cuff around the thigh. The VAR was recorded at the cuff pressures of 30 mmHg and 60 mmHg. Ten patients with chronic venous insufficiency (CVI) and 20 control subjects with healthy legs were investigated. The VAR increased in relation to the increase of cuff pressure at both wavelengths. There were no significant differences in the VAR between the cuff pressures within or between the legs with CVI and healthy legs. The VAR measured at 780 nm was very significantly greater than the VAR measured at 543 nm in legs with CVI (p<0.005), as well as in healthy legs (p<0.001). The VAR depends both on the wavelength of the laser Doppler light used and on the degree of venous hypertension. The VAR is not impaired in legs with CVI compared with healthy legs.

Increased plasma vascular endothelial growth factor among patients with chronic venous disease

Skin damage in the presence of chronic venous disease is partially mediated through leukocytes. The endothelium is activated and exhibits proliferation in the skin. Up-regulation of vascular endothelial growth factor (VEGF) expression in the skin of patients with chronic venous disease has been demonstrated with immunohistologic techniques. Abnormal VEGF expression can have local deleterious effects. The aim of this study was to determine whether patients with chronic venous disease have elevated plasma levels of VEGF.We conducted a prospective study with 30 patients with varicose veins of clinical, etiologic, anatomic, and pathologic class C3 (normal skin, n = 15) and C4 (trophic skin changes, n = 15) and 25 control subjects with no clinical evidence of venous or arterial disease of the lower limb. Blood samples were collected from a foot vein of each subject before and after a period of experimental venous hypertension produced by means of standing. Assay of VEGF protein was performed with a sandwich enzyme-linked immunosorbent assay.Plasma VEGF level was elevated in both groups of patients with venous disease compared with the control group. The median VEGF levels among patients were 81 pg/mL (interquartile range [IQR] 56 to 122) supine and 98 pg/mL (IQR 63 to 153) after standing for 30 minutes. Median VEGF levels among control subjects were 52 pg/mL (IQR 35 to 71) lying supine and 60 pg/mL (IQR 39 to 105) after standing for 30 minutes. Experimental venous hypertension caused a small rise in VEGF levels among the patients but not the control subjects. Further studies are required to determine whether increased VEGF expression contributes to tissue injury in chronic venous disease. (J Vasc Surg 1998;28:535-40.)

Endothelial activation in patients with chronic venous disease

Leukocyte trapping due to leukocyte-endothelial activation has been implicated as the cause of lipodermatosclerosis and ulceration in patients with chronic venous disease. We investigated endothelial activity in normal controls and patients subjected to short-term venous hypertension. Twenty-five normal volunteers and 30 patients with chronic venous disease divided into two groups: varicose veins with skin changes (LDS, n = 15); and varicose veins without skin changes (VVs, n = 15) were studied. Blood samples were taken from a foot vein before and after experimental venous hypertension. Plasma levels of ELAM-1 (endothelial leukocyte adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), and von Willebrand factor (vWf) was measured by an ELISA. There was a significant rise in the plasma concentration of ELAM-1, ICAM-1 and VCAM-1 in patients and normal controls in response to venous hypertension. Basal levels of plasma VCAM-1 and vWf were higher in patients with LDS compared to patients with VVs. The magnitude of rise of VCAM-1 was greater in patients with LDS compared to patients with VVs (p = 0.01, Mann-Whitney U-test). There was no difference in the basal levels or in the magnitude of change in plasma ICAM-1 and ELAM-1 between the two patient groups. Venous hypertension results in endothelial activation which may aid endothelial-leukocyte adhesion. Patients with LDS exhibit increased VCAM-1, which is a counterligand for receptors expressed by monocytes and lymphocytes signifying that these cells may be more important in the development of skin changes.

Leucocyte Activity in Volunteers in Response to Experimental Venous Hypertension

Objective: To investigate leucocyte activation in normal volunteers subjected to short-term venous hypertension by measuring the cell surface expression of the integrin CD11b and the selectin CD62L on neutrophils and monocytes before and after short-term venous hypertension. Plasma levels of L-selectin, which is shed by leucocytes upon activation, were also measured. Design: Prospective study, measuring white cell count, neutrophil and monocyte surface CD11b and L-selectin expression in whole blood using a fluorescent-labelled monoclonal antibody in a flow cytometer, and plasma L-selectin by a commercially available ELISA. Setting: The Middlesex Hospital Vascular Laboratory, a referral centre for the investigation of venous diseases. Subjects: Twenty-five normal volunteers without any history or clinical finding of venous disease. Results: There was a significant fall in the white cell: red cell ratio ( p = 0.03, Wilcoxon) in response to venous hypertension, followed by a significant increase ( p &lt; 0.001, Wilcoxon) when the venous hypertension was reversed. Neutrophil and monocyte CD11b levels fell significantly ( p &lt; 0.001 and p &lt; 0.001, Wilcoxon) in response to venous hypertension. On reversing the venous hypertension a significant increase of CD11b expression was noted in both neutrophils and monocytes ( p &lt; 0.001 and p &lt; 0.01, Wilcoxon). Surface L-selectin expression, however, showed a significant sustained fall in both neutrophils and monocytes (neutrophils, p = 0.05 and p = 0.02; monocytes, p = 0.04 and p = n.s., Wilcoxon) throughout the experiment. There was a significant rise in soluble L-selectin in the plasma following venous hypertension ( p &lt; 0.001, Wilcoxon). Conclusion: This study demonstrates both neutrophil and monocyte activation in the microcirculation of normal volunteers in response to short-term venous hypertension.

Leukocyte migration in the leg in response to experimental venous hypertension.

Leukocyte trapping and activation in the microcirculation of the legs may play an important role in causing skin damage in venous disease. Leukocyte emigration from the microcirculation and subsequent locomotion in response to venous hypertension was studied in a group of 12 normal volunteers using a "skin window" technique. Two 0.5-cm square dermal abrasions were made with a dental stone over the gaiter area of the leg and the flexor aspect of the forearm (control), which were covered with moist micropore membranes. The volunteers lay supine for 30 minutes, and then stood supported for 30 minutes to raise the venous pressure in the leg, and then lay supine again for 30 minutes. The experiment was repeated in six volunteers who lay supine for the whole period. The membranes were changed and collected every 15 minutes, fixed in formal saline solution, and dual-stained for monocytes and polymorphonuclear leukocytes. The type and numbers of cells that emigrated and the furthest distance traveled (leading front) by the cells through the membrane were measured. Both in arms and legs, the vast majority of cells that emigrated were neutrophils, with very few monocytes (arm, 93% neutrophils and 7% monocytes; leg, 97% neutrophils and 3% monocytes). In the 30 minutes after venous hypertension, leukocyte migration significantly decreased in the leg (median leukocyte locomotion: basal, 75.3 microns; standing, 73.5 microns; after hypertension, 62.9 microns; p = 0.012, Wilcoxon matched pairs signed rank test), but not in the arm (basal, 86.2 microns; standing, 84.4 microns; after hypertension, 85.5 microns; p = NS) or when the experiment was repeated with the volunteers lying supine for the entire period (basal, 91.5 microns; standing, 89.4 microns; after hypertension, 92.6 microns; p = NS). Leukocyte migration is decreased immediately after experimental venous hypertension, which may be a result of the release of factors that inhibit migration.

Neutrophil Activation in Experimental Venous Hypertension

Objective: To investigate the white cell trapping hypothesis of venous ulceration by measuring plasma lactoferrin as a marker of neutrophil degranulation in normal volunteers in two experimental models of venous hypertension. Design: A prospective study of volunteers with no history or clinical evidence of venous disease. Setting: The Middlesex Hospital Vascular Laboratory, Mortimer Street, London WIN 8AA, UK. Patients: Volunteers within the Middlesex Hospital Vascular Laboratory with no history or clinical findings of venous or arterial disease, no other systemic disease, on no medication known to alter white cell activity, and with no recent infection. Interventions: Venous blood was taken from cannulae in both feet and the right arm for a neutrophil count and Plasma lactoferrin, measured using an ELISA, during application of a tourniquet to 80 mmHG for 30 min to the right leg while supine, 5 min after release of tourniquet, and then during a 30 min period of standing. Results: During application of a tourniquet to the right leg there was a significant rise in plasma lactoferrin and in lactoferrin corrected for the neutrophil count ( p &lt; 0.05, Wilcoxon). In the unoccluded leg, although Plasma lactoferrin rose, this was not significant when corrected for the rise in neutrophil count. After standing for 30 min, the lactoferrin and neutrophil count increased in all three limbs; corrected lactoferrin showed a significant increase in the legs ( p &lt; 0.02), though not in the arm. Conclusion: Increased neutrolphil degranulation occurs during periods of short-term venous hypertension in normal volunteers, in keeping with the white cell trapping hypothesis.

Microcirculatory aspects of venous ulceration.

Articles were identified from a literature search based on Index Medicus from 1983 onwards with additional, as yet, unpublished data obtained directly from ongoing research at The Middlesex Hospital Vascular Laboratory. Original scientific articles were selected if they addressed the mechanisms causing venous ulceration in relation to the events in the microcirculation. To review the mechanisms proposed recently to explain the pathogenesis of venous ulceration. Recent data suggest that there is a systemic inflammatory response associated with the presence of venous disease in the lower limb, as indicated by measuring plasma neutrophil elastase and lactoferrin. In volunteers without venous disease white cell activation may be produced by experimental venous hypertension lasting 30 minutes, produced by applying a venous tourniquet to the lower limb or by standing without moving for 30 minutes. Inflammatory mechanisms play a significant role in the pathogenesis of venous disease but the exact mechanisms producing venous ulceration remain to be elucidated.

Effects of portal vein stenosis and superior mesenteric vein ligation on mesenteric venous pressure and porta-systemic shunting in the rat.

We studied the relative contributions of splanchnic congestion and porta-systemic shunting to the maintenance of experimental portal venous hypertension. Three groups of rats were prepared: portal vein-stenosed, superior mesenteric vein-ligated and sham operated. Though elevated in both operated groups compared to controls, mesenteric venous pressure was highest in the portal vein-stenosed animals (PV vs SMV vs Sham: 19.6 +/- 1.3 vs 15.6 +/- 0.7 vs 13 +/- 0.6; p < .05 PV and SMV vs Sham, and PV vs SMV) despite the presence of 50% porta-systemic shunting in the portal vein-stenosed animals. Shunting was negligible in the other two groups. Peripheral plasma glucagon and vasoactive intestinal peptide (VIP) levels were similar in all three groups. We conclude that mesenteric congestion alone plays a minor role in the pathogenesis of portal hypertension, which may instead be related to the porta-systemic shunting of vasoactive substances other than glucagon and VIP.