Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells.

The objective of this study was to assess the effects of two structurally distinct yet selective proteasome inhibitors (PS-341 and lactacystin) on leukocyte adhesion, endothelial cell adhesion molecule (ECAM) expression, and nuclear factor-kappaB (NF-kappaB) activation in tumor necrosis factor (TNF)-alpha-stimulated human umbilical vein endothelial cells (HUVEC) and the transformed, HUVEC-derived, ECV cell line. We found that TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclear neutrophil (PMN) adhesion to HUVEC but not to ECV; TNF also significantly enhanced surface expression of vascular cell adhesion molecule 1 and E-selectin (in HUVEC only), as well as intercellular adhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVEC with lactacystin completely blocked TNF-stimulated PMN adhesion, partially blocked U-937 adhesion, and completely blocked TNF-stimulated ECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1 expression in ECV. Pretreatment of HUVEC with PS-341 partially blocked TNF-stimulated leukocyte adhesion and ECAM expression. These effects of lactacystin and PS-341 were associated with inhibitory effects on TNF-stimulated NF-kappaB activation in both HUVEC and ECV. Our results demonstrate the importance of the 26S proteasome in TNF-induced activation of NF-kappaB, ECAM expression, and leukocyte-endothelial adhesive interactions in vitro.

Differential monocyte adhesion and adhesion molecule expression in venous and arterial endothelial cells.

We compared U-937 cell adhesion and adhesion molecule expression in human umbilical venous (HUVECs) and arterial (HUAECs) endothelial cells exposed to tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide (LPS). TNF and LPS stimulated vascular cell adhesion molecule (VCAM)-1 surface expression and adhesion of U-937 monocyte-like cells to HUVECs but not to HUAECs. Antibody studies demonstrated that in HUVECs at least 75% of the adhesion response is VCAM-1 mediated. Interleukin-1 stimulated U-937 cell adhesion to and VCAM-1 surface expression in both HUVECs and HUAECs. Pyrrolidinedithiocarbamate and the proteasome inhibitor MG-132 blocked TNF- and LPS-stimulated U-937 cell adhesion to HUVECs. These agents also significantly decreased TNF- and LPS-stimulated increases in HUVEC surface VCAM-1. TNF increased VCAM-1 protein and mRNA in HUVECs that was blocked by pyrrolidinedithiocarbamate. However, neither TNF or LPS stimulated VCAM-1 expression in HUAECs. TNF stimulated expression of both intercellular adhesion molecule-1 and E-selectin in HUVECs, but in HUAECs, only intercellular adhesion molecule-1 was increased. Electrophoretic mobility shift assays demonstrated no difference in the pattern of TNF-stimulated nuclear factor-kappaB activation between HUVECs and HUAECs. These studies demonstrate a novel and striking insensitivity of arterial endothelium to the effects of TNF and LPS and indicate a dissociation between the ability of HUAECs to upregulate nuclear factor-kappaB and VCAM-1.

Ultrasound densitometric analysis of carotid plaque composition. Pathoanatomic correlation.

BACKGROUND AND PURPOSE: The components of a carotid artery plaque might affect the risk of ipsilateral stroke. The accuracy of carotid duplex scan in assessing stroke risk reflects the experience of the scan reader. Thus, methods that can enhance ultrasonic evaluation of plaque morphology might allow a more objective means of determining carotid-mediated stroke risk. METHODS: We performed densitometric analysis of B-mode images of carotid plaques in nine patients scheduled for carotid endarterectomy. All patients had preoperative duplex color imaging and cerebral arteriography. The surgical specimen was analyzed histologically to determine the plaque components (soft plaque/organized thrombus, intraplaque hemorrhage/lipid deposition, fibrosis, and calcification). The specimen findings were correlated with the densitometric measurements to determine whether the density analysis would allow a reliable determination of the plaque substratum. RESULTS: With 1.0 as a reference point for the moving column of blood, the mean acoustic densities (+/-SD) were as follows: organized thrombus, 1.8 +/- 0.5; intraplaque hemorrhage/lipid deposition, 5.15 +/- 0.9; fibrosis, 9.51 +/- 2.9; and calcification, 15.5 +/- 8.6. CONCLUSIONS: We conclude that densitometric evaluation allows differentiation of the various possible components of carotid plaque. The determination of plaque composition, based on density measurement, may provide information about its potential for thromboembolization.

Intermittent pneumatic compression improves venous ulcer healing.

The effects of intermittent pneumatic compression on the healing rates of ulcers in patients with chronic venous insufficiency were examined in a prospective, controlled study of 22 patients. Patients were randomly assigned to the experimental or the control group. Both groups received local wound care followed by application of an Unna boot. In addition, subjects in the experimental group received intermittent pneumatic compression (IPC) twice weekly for one hour each session. Healing rates were reported in square centimeters per day. Data analysis revealed a mean healing rate of 0.08 cm2 per day for control subjects and 0.15 cm2 per day for experimental subjects. Statistical analysis, demonstrated the healing rates of the two groups to be statistically different. The results appear to indicate that intermittent pneumatic compression is beneficial in the management of venous insufficiency ulcers.

Hemodynamic evaluation of a bioprosthetic venous prosthesis.

PURPOSE: A prosthetic venous valve must be biocompatible and nonthrombogenic and function in the venous circulation. Biocompatibility and thrombogenicity of our prosthesis have been examined in prior animal experiments, and 91% of valve conduits including early prototypes are patent at 3 weeks. However, evaluation of valve function is much more difficult in animals; therefore in this study the function of excised valves was evaluated ex vivo. METHODS: Nine bovine jugular vein conduits, each with one bileaflet venous valve, were harvested and placed in a venous flow simulator. Flows and pressures were adjusted to mimic human respiratory and hydrostatic variations. Each valve and conduit was tested for variations in valve diameter and sinus expansion in response to flow. Valve opening and closing times and valve competence were measured in response to pressure changes. After testing, each specimen was glutaraldehyde fixed and assessed a second time. RESULTS: Valve orifice area increased in response to flow in both fresh and fixed tissues. Maximum valve orifice area was reduced by fixation (27.7%) at full flows (p < 0.05). Valve sinus dimensions increased in response to increased pressure until maximum expansion was achieved (33 mm Hg). This was reduced 15.3% in fixed tissue (p < 0.05). Valve opening times (at < 1 mm Hg gradient) were slightly longer in fixed compared with fresh tissue (0.43 +/- 0.09 vs 0.41 +/- 0.13 second; p < 0.05). Valve closing times were comparable in both states (0.43 +/- 0.08 vs 0.49 +/- 0.07 second). Three fresh and seven fixed specimens that were subjected to 287 mm Hg back pressure exhibited minimal reflux. CONCLUSIONS: Size and availability make the bovine jugular vein valve an ideal venous valve substitute. Glutaraldehyde fixation renders the tissue biocompatible and nonthrombogenic while preserving anatomic integrity and leaflet strength and flexibility. Mounted and stented in a sewing sleeve, this prosthesis could represent the first generally applicable clinical solution to chronic venous insufficiency and venous hypertension.

In vitro performance of venous valve prostheses. An experimental model study.

The performance of fresh and glutaraldehyde fixed bovine jugular vein valves (10 mm diameter) was investigated in an experimental flow loop that provides adjustable flow rates and a downstream oscillatory pressure. Three different venous valve (VV) conduit geometries (curved [C], straight [S], and tapered [T]), were tested. The flow loop consisted of two independently adjustable components, with the mean flow generated by adjusting the elevation difference between the head tank and outflow chamber. An adjustable sinusoidal pressure pulse was superimposed on the downstream of a VV to mimic the respiratory effects. Flow visualizations were made using 100 microns mica chip tracers in the laser illuminated flow fields. To assess VV performance under various flow conditions, the closure opening (CO), partial opening (PO), and leaflet fluttering (LF) were evaluated. At a given pulse pressure, the three conduits required different flow rates to reach CO mode. At 12 cm H2O pulse pressure, the fresh valve in C-conduit exhibited stable CO operation at a flow rate of 1.01 ml/sec. That in S and T conduits required 1.67 ml/sec and 2.25 ml/sec, respectively. At higher flow rates, PO and LF performances were observed in all three conduits. Different threshold values of pulse pressure were needed to reestablish the CO operation mode for the C, S, and T conduits, individually. These observations provide some insight into the role of conduit geometry and sinus configuration in the function of VV.

The residual central venous catheter track--an occult source of lethal air embolism: case report.

Air embolization through a residual track after removal of a central venous catheter is an elusive mechanism of a treacherous problem. Prompt diagnosis is necessary for successful application of established therapy, as demonstrated by this case report. Awareness of this unusual mechanism of air embolism with utilization of simple prophylactic measures, however, remains the best treatment.

In vitro testing of venous valves.

The need to test prosthetic venous valves led to the design of a hydraulic mock circuit that reliably mimics natural venous flow. Components of this system simulate calf muscle pump and thoracoabdominal suction pump action. Flow and pressures are serially measured along the circuit. Valve function can be observed and videotaped. Evaluation of biologic venous valves provides visual and quantitative assessment of venous valve function with respect to tissue processing, valve design, and implantation technique. Further improvement in this system in terms of more compliance and less rigidity of components is being pursued.

Occult antithrombin III deficiency: a potentially lethal complication of the postphlebitic limb.

Chronic venous insufficiency is a frequent sequel to lower extremity venous thrombosis. A relatively uncommon, but potentially lethal, cause of the thrombosis is congenital antithrombin III deficiency. Recognition and treatment of this occult deficiency is critical. The following report describes a family treated by the authors for this problem. In one generation of nine siblings, three males had documentation of the disease with functional antithrombin III levels in the range of 50% to 60%. Before evaluation for the deficiency one female sibling died at the age of 20 years as a consequence of a proven pulmonary embolus. Antithrombin III levels in another female sibling, who was free of symptoms, were normal (80% to 120%). Four other siblings who were free of symptoms (one female, three males) refused evaluation. All three men with the deficiency had severe, chronic, bilateral, lower extremity, venous insufficiency manifested by pain, varicosities, edema, pigmentation, and ulceration. Despite chronic warfarin therapy, one experienced recurrent pulmonary embolization with eventual loss of perfusion of the entire right lung. Ascending venography in the symptomatic males with the deficiency revealed evidence of recurrent and diffuse venous thrombosis with partial recanalization. Recurrent lower extremity venous thrombosis consequent to antithrombin III deficiency causes a particularly fulminant postphlebitic syndrome with characteristic venographic findings. Although potentially lethal if unrecognized and treated simply as venous insufficiency, chronic therapy with warfarin offers palliation and prolongs life.

Valvular xenografts in the inferior vena cava.

The unsolved problem of valvular incompetence is a common denominator to chronic venous insufficiency. Cardiac experience with tissue valves suggests the potential for their use in more peripheral locations. The current initial work evaluated 22 valvular xenografts placed in the inferior vena cavas of 22 dogs. Patency extended to 28 months in 12 valves. There was a plausible explanation for failure in 8 of the 10 unsuccessful units. Patent valves functioned with minimal pressure gradients at moderate flow with no evidence of pulmonary emboli. Retrograde valvulograms showed acceptable functional competence in six of eight units examined despite autopsy evidence of leaflet fibrosis and anatomic incompetence. The study establishes the feasibility of long-term patency of valvular xenografts in the inferior vena cava, but also raises questions concerning design requirements for prosthetic venous valves.

Gastrojejunal fistula: a complication of peptic ulcer disease.

Spontaneous gastrojejunal fistula formation is rare and its differential diagnosis multifactorial. Precise etiologic determination is necessary for proper management. We have reported a case typical of gastrojejunal fistula due to peptic ulcer disease. In contrast to the gastrocolic fistula, symptoms of the gastrojejunal fistula are those of the ulcer diathesis itself. The preferred management is by en bloc one-stage resection after preoperative colon preparation.

Renal acid-base metabolism after ischemia.

The response of the kidney to ischemia-induced cellular acidosis was followed over the immediate one hr post-ischemia reflow period. Clearance and extraction experiments as well as measurement of cortical intracellular pH (pHi) were performed on Inactin-anesthetized Sprague-Dawley rats. Arteriovenous concentration differences and para-aminohippurate extraction were obtained by cannulating the left renal vein. Base production was monitored as bicarbonate released into the renal vein and urine; net base production was related to the renal handling of glutamine and ammonia as well as to renal oxygen consumption and pHi. After a 15 min control period, the left renal artery was snared for one-half hr followed by release and four consecutive 15 min reflow periods. During the control period, cortical cell pHi measured by [14C]-5,5-Dimethyl-2,4-Oxazolidinedione distribution was 7.07 +/- 0.08, and Q-O2 was 14.1 +/- 2.2 micromoles/min; neither net glutamine utilization nor net bicarbonate generation occurred. After 30 min of ischemia, renal tissue pH fell to 6.6 +/- 0.15. However, within 45 min of reflow, cortical cell pH returned and exceeded the control value, 7.33 +/- 0.06 vs. 7.15 +/- 0.08. This increase in pHi was associated with a significant rise in cellular metabolic rate, Q-O2 increased to 20.3 +/- 6.4 micromoles/min. Corresponding with cellular alkalosis was a net production of bicarbonate and a net ammonia uptake and glutamine release; urinary acidification was abolished. These results are consistent with a nonexcretory renal metabolic base generating mechanism governing cellular acid base homeostasis following ischemia.

Long-term patency of venous repairs demonstrated by venography.

Indications for venous reconstruction after traumatic injury are controversial, partly because of uncertainty of continued patency. We found no reports in the literature of truly long-term results after venous reconstruction. For clarification of this issue, we reviewed femoral venous injuries in a civilian metropolital population over a 20-year period. There were 31 patients with penetrating femoral venous injuries. Twenty-four patients underwent reconstruction. A search for these patients years after reconstruction located only five patients with six reconstructions. Followup venography at 6 to 20 years demonstrated venous patency and functional valves with asymptomatic patients and no clinical evidence of venous insufficiency in all cases excluding a single 1962 repair with a Teflon graft. This study then supports reconstruction rather than ligation after venous trauma. Although small, this series appears to be the only known report of truly long-term results following venous reconstruction.

Venous prosthetic valves. The first step toward an investigation in the canine model.

Bioprosthetic valves were surgically placed in the inferior vena cava of four canines as a first step in the investigation of prosthetic valves for the treatment of nonobstructive chronic venous stasis. Valve patency and thrombus formation involving either the valve or the IVC was evaluated by serial follow-up inferior vena cavagrams in each canine. No thrombus occurred in any of the canines as observed at six months in two canines and eight months in two canines. The ultimate goal of this and other ongoing investigations is to develop a valvular prosthesis which may be placed into the venous system for the management of nonobstructive chronic venous stasis.

Amputation risk factors in concomitant superficial femoral artery and vein injuries.

Only a small subset of patients with combined superficial femoral artery and vein injuries results in amputation. The importance of the venous component as a risk factor for amputation is uncertain. Ligation vs. reconstruction of venous injuries is controversial. For clarification of these issues, we analyzed retrospectively multiple risk factors for amputation in combined superficial femoral artery and vein injuries in a civilian population. There were 25 patients treated in a 20-year period. Sixteen injuries were caused by small caliber missiles, six by shotgun blasts, and three by knife wounds. Three patients (12%) ultimately underwent amputation. The major risk factor for amputation was method of vascular reconstruction. All three amputations underwent ligation of the superficial femoral vein with arterial reconstruction by placement of a reversed interposition saphenous vein graft (p = 0.0009). None of the remaining 22 patients with salvaged limbs underwent reconstruction by this combination of techniques. Consequently, the authors emphasize the importance of venous reconstruction, particularly in combined injuries with major arterial involvement requiring interposed grafts.

Gamma-glutamyltransferase release by the post ischemic kidney: multiple forms and cellular pH.

The release of gamma-glutamyltransferase from renal tubule cells was studied in situ following 30 minutes of ischemia. The ischemic kidney enzyme level fell 33 percent after 15 minutes of reflow of which only 1.2 percent was recovered in the urine; none was released into the renal vein. At this time the overwhelming majority of the enzyme appears bound to membranes in both the kidney and the urine. In the subsequent 15 minutes renal levels continue to decline while urinary excretion accounts for 5 percent of that disappearing from the kidney. Interestingly the form of the enzyme present in kidney and urine shifts to a soluble form coinciding with cellular alkalosis, urinary alkalinization and a rise in ATP levels. Alkalinization of renal homogenates result in a 2-fold increase in the soluble enzyme form. The results are consonant with the immediate loss of brush border enzyme via uptake into the cell or release into the urine with the former pathway predominating; subsequent appearance of the soluble enzyme appears to reflect intracellular alkaline proteinase activity and exocytosis. The form in which the enzyme is excreted may provide a useful clinical index: membranous reflecting cellular necrosis and soluble reflecting cellular recovery.