Rajabrata Sarkar

Nitric Oxide Reversibly Inhibits the Migration of Cultured Vascular Smooth Muscle Cells

Augmentation of nitric oxide (NO) production in vivo decreases lesions in a variety of models of arterial injury, and inhibition of NO synthase exacerbates experimental intimal lesions. Both vascular smooth muscle cell (VSMC) proliferation and migration contribute to lesion formation. Although NO inhibits VSMC proliferation, its effects on VSMC migration are unknown. To test the hypothesis that NO inhibits VSMC migration independent of inhibition of proliferation, we examined migration of rat aortic VSMCs after wounding of a confluent culture in the presence of chemical donors of NO. Hydroxyurea was used to eliminate any confounding effect of NO on proliferation. Three NO donors, diethylamine NONOate, spermine NONOate, and S-nitrosoglutathione, exhibited concentration-dependent inhibition of both number of migrating VSMCs and maximal distance migrated. Inhibition of migration was also seen with 8-Br-cGMP, suggesting that activation of guanylate cyclase may play a role in mediating the antimigratory effects of NO. Migration resumed after removal of NO donors, as evidenced by an increase in distance migrated. Measurement of VSMC protein synthesis and mitochondrial respiration indicated that inhibition of migration by NO donors was not due to metabolic cytostasis. These findings indicate that NO reversibly inhibits VSMC migration independent of proliferation or cytotoxicity, a novel mechanism by which both endogenous and pharmacological NO may alter vascular pathology.

Prosthetic replacement of the inferior vena cava for malignancy

Abstract Purpose: Invasion of the inferior vena cava (IVC) by tumor is generally considered a criterion of unresectability. This study was designed to review the outcomes of a strategy of aggressive resection of the vena cava to achieve complete tumor resection coupled with prosthetic graft placement to re-establish caval flow. Methods: Retrospective review of patients treated at a university referral center. Ten patients (mean age 54; eight females, two males) underwent tumor resection that involved circumferential resection of the IVC and immediate prosthetic replacement with ringed polytetrafluoroethylene (PTFE) grafts ranging in diameter from 12 to 16 mm. Results: Seven patients had replacement of the infrarenal IVC, two of their suprarenal IVC, and one had reconstruction of the IVC bifurcation. Four of the 10 patients received preoperative chemotherapy, and none received radiotherapy. The most common (7/10) pathologic diagnosis was leiomyosarcoma arising from the IVC or retroperitoneum. Additional diagnoses included teratoma (one), renal cell carcinoma (one), and adrenal lymphoma (one). There were no perioperative deaths, and one complication (prolonged ileus) occurred. Mean length of stay was 8.1 days. Anticoagulation was not routinely used intraoperatively or postoperatively. Follow-up (mean duration=19 months) demonstrated that survival was 80% (8/10) and 88% (7/8) of patients were free of venous obstructive symptoms. Conclusion: Resection of the IVC with prosthetic reconstruction allows for complete tumor resection and provides durable relief from symptoms of venous obstruction. (J Vasc Surg 1998;28:75-83.)

Arterial aneurysms in children: Clinicopathologic classification

Thirty-one arterial macroaneurysms in 23 pediatric-aged patients (16 boys and 7 girls) were treated at the University of Michigan. The average age at time of diagnosis was 10.2 years (range 6 months to 18 years). Vessels involved the aorta (4), as well as hepatic (1), splenic (2), gastroepiploic (1), renal (12), iliac (1), superficial femoral (4), popliteal (1), brachial (1), radial (2), and ulnar (2) arteries. Twelve children exhibited overt clinical manifestations including presence of a mass (7), local pain (3), hematemesis (1), and painless obstructive jaundice (1). Eleven children had asymptomatic lesions. Aneurysm existence was confirmed by arteriography or operation. All but one child underwent surgical therapy, with 20 long-term survivors (mean follow-up 3.5 years). One operative death occurred and one death occurred 6 years after surgery. This experience and a review of previously reported cases served as a basis for categorization of childhood aneurysmal disease as true aneurysms associated with (I) arterial infection, (II) giant-cell aortoarteritis, (III) autoimmune connective tissue disease, (IV) Kawasakis disease, (V) Ehlers-Danlos syndrome or Marfans syndrome, (VI) other forms of noninflammatory medial degeneration, (VII) arterial dysplasias, (VIII) congenital-idiopathic factors, as well as (IX) false aneurysms associated with extravascular events causing vessel wall injury or disruption. Knowledge of the varied clinicopathologic characteristics of arterial aneurysms in children is important in treating these patients.

Transforming growth factor-β1 increases lysyl oxidase enzyme activity and mRNA in rat aortic smooth muscle cells

Abstract Purpose: This investigation was designed to test the hypothesis that transforming growth factor-β 1 (TGF-β 1 ) regulates lysyl oxidase secretion from vascular smooth muscle cells. Lysyl oxidase is an enzyme that catalyzes an essential step in collagen and elastin cross-linking in the extracellular matrix, and TGF-β 1 has been implicated in the pathogenesis of restenosis after vascular injury. The effect of TGF-β 1 on lysyl oxidase in vascular smooth muscle cells has not been previously defined. Methods: Rat aortic smooth muscle cells were grown in culture to confluence. Cells in passage 2 to 6 were incubated for 24 hours in media containing 0.1, 0.5, 1.0, or 10.0 ng/ml of TGF-β 1 . Lysyl oxidase activity in the media was quantitated with a tritium-release bioassay against an insoluble 3 H-labeled aortic elastin substrate. Northern blot analyses were performed to determine steady-state levels of lysyl oxidase mRNA in the smooth muscle cells. Results: Lysyl oxidase activity in the media increased 1.5-fold above control levels after exposure to 10 ng/ml of TGF-β 1 ( p 1 , respectively ( p 1 was also time-dependent over the 24-hour experimental period. Conclusions: TGF-β 1 appears to regulate lysyl oxidase in cultured rat aortic smooth muscle cells. Increases in lysyl oxidase activity may be one of the mechanisms by which TGF-β 1 contributes to arterial restenosis after vascular injury. (J Vasc Surg 1997;25:446-52.)

Surgical approach to cecal diverticulitis

BACKGROUND Cecal diverticulitis is a rare condition in the Western world, with a higher incidence in people of Asian descent. The treatment for cecal diverticulitis has ranged from expectant medical management, which is similar to uncomplicated left-sided diverticulitis, to right hemicolectomy. STUDY DESIGN A retrospective chart review was conducted of the 49 patients treated for cecal diverticulitis at Olive View-UCLA Medical Center from 1976 to 1998. This was the largest-ever single-institution review of cecal diverticulitis reported in the mainland US. RESULTS The clinical presentation was similar to that of acute appendicitis, with abdominal pain, low-grade fever, nausea/vomiting, abdominal tenderness, and leukocytosis. Operations performed included right hemicolectomy in 39 patients (80%), diverticulectomy in 7 patients (14%), and appendectomy with drainage of intraabdominal abscess in 3 patients (6%). Of the 7 patients who had diverticulectomy, 1 required right hemicolectomy at 6 months followup for continued symptoms. Of the three patients who underwent appendectomy with drainage, all required subsequent hemicolectomy for continued inflammation. Of the 39 patients who received immediate hemicolectomies, there were complications in 7 (18%), with no mortality. CONCLUSIONS We endorse an aggressive operative approach to the management of cecal diverticulitis, with the resection of all clinically apparent disease at the time of the initial operation. In cases of a solitary diverticulum, we recommend the use of diverticulectomy when it is technically feasible. When confronted with multiple diverticuli and cecal phlegmon, or when neoplastic disease cannot be excluded, we advocate immediate right hemicolectomy. This procedure can be safely performed in the unprepared colon with few complications. Excisional treatment for cecal diverticulitis prevents the recurrence of symptoms, which may be more common in the Western population.

Nitric oxide inhibition of endothelial cell mitogenesis and proliferation

BACKGROUND Endothelial cell (EC) proliferation is essential in vascular repair after injury to the vessel wall. Impaired EC proliferation may be an important factor contributing to vessel wall disease. Nitric oxide (NO) inhibits proliferation of many cells, including smooth muscle cells (SMC). We tested the hypothesis that NO inhibits EC proliferation and DNA synthesis. METHODS Cultured canine venous ECs were treated with NO donors: S-nitroso-N-acetylpenicillamine (SNAP), S-nitroso-glutathione (GSNO), or spermine NONOate (SP NO). Proliferation was determined by cell counts after 48 hours. Parallel proliferation studies were done with rat aortic SMC. ECs synchronized in S phase were treated with the NO donor diethylamine NONOate (DEA NO), and DNA synthesis was measured as the incorporation of tritiated thymidine. A NO antagonist, cPTIO, was used to reverse the effects of DEA NO: RESULTS Concentration-dependent (1 to 100 mmol/L) inhibition of EC proliferation (11% to 71% inhibition; p < 0.05) was seen with SNAP. Similar inhibition of proliferation was noted with the NO donors GSNO and SP NO and in SMC treated with SNAP. DEA NO caused concentration-dependent (0.1 to 1 mmol/L) inhibition of EC DNA synthesis (39% to 85% inhibition; p < 0.05), which was reversed by cPTIO. CONCLUSIONS NO inhibits proliferation and mitogenesis of cultured ECs. This may occur in certain pathologic states, where production of NO in plaques and diseased vessels impedes reendothelialization, thus contributing to adverse thrombotic and vasospastic events.

Dual cell cycle-specific mechanisms mediate the antimitogenic effects of nitric oxide in vascular smooth muscle cells

Objective To determine the cell cycle specificity and intracellular mechanisms involved in inhibition by nitric oxide (NO) of vascular smooth muscle cell mitogenesis. Methods Cultured rat aortic smooth muscle cells were synchronized by serum withdrawal, treated with the NO donor S-nitroso-N-acetylpenicillamine and the cyclic GMP analog 8-Br-cGMP at various times during cell cycle progression, and DNA synthesis measured during the S phase. Two additional NO donors, 5-nitroso-glutathione and diethylamine NONOate, were used to confirm the inhibition of DNA synthesis by S-nitroso-N-acetylpenicillamine, and the ability of two antagonists of free NO to reverse the effects of NO donors was also evaluated. Bypass of ribonucleotide reductase by use of exogenous deoxynucleosides was attempted to determine whether inhibition of this S-phase enzyme was the mechanism by which NO inhibited DNA synthesis during the S phase. Results Vascular smooth muscle cell mitogenesis was inhibited by cyclic GMP (cGMP) up to late G1 phase of the cell cycle, which corresponded to the point of greatest sensitivity to exogenous NO. In contrast to cGMP, three different NO donors inhibited DNA synthesis when added to cells synchronized in S phase, beyond the restriction point of cell cycle control in late G1 phase. This S-phase inhibition was reversible by removal of the NO donor or addition of two NO antagonists and was not observed with non-NO analogs of the donors. Inhibition by NO donors in S phase was neither reversed by the guanylate cyclase inhibitor methylene blue nor mimicked by exogenous cGMP. The S-phase inhibition by all three NO donors was reversed partially by bypass of ribonucleotide reductase, establishing this enzyme as an S-phase target of NO. Conclusions These findings demonstrate that NO inhibits smooth muscle mitogenesis by cGMP-dependent and -independent mechanisms acting at distinct points in the cell cycle. NO is the first endogenous substance to have been shown to inhibit mitogenesis beyond the restriction point in late G1 phase, suggesting that it plays a role in regulation of cells that have lost normal mechanisms of G1 growth control, such as the hyperproliferative smooth muscle cells noted in hypertension and restenosis.

Adeno-associated viral vector-mediated gene transfer of VEGF normalizes skeletal muscle oxygen tension and induces arteriogenesis in ischemic rat hindlimb

Critical limb ischemia is an important clinical problem that often leads to disability and limb loss. Vascular endothelial growth factor (VEGF), delivered either as recombinant protein or as gene therapy, has been shown to promote both collateral artery formation (arteriogenesis) and capillary angiogenesis in animal models of hindlimb ischemia. However, none of the previous studies has demonstrated an improvement in tissue hypoxia, the condition that drives the molecular response to ischemia. Furthermore, the optimal vector and route of gene delivery have not been determined. Recently, adeno-associated viral (AAV) vectors, which efficiently transduce skeletal muscle and produce sustained transgene expression, have been used as gene therapy vectors. We asked whether an intra-arterial injection of AAV-VEGF(165) normalizes muscle oxygen tension by increasing skeletal muscle oxygen tension, and promotes arteriogenesis and angiogenesis in a rat model of severe hindlimb ischemia. We found that AAV-VEGF treatment normalized muscle oxygen tension in the ischemic limb. In contrast, vehicle and AAV-lacZ-treated limbs remained ischemic. Collateral arteries were more numerous in AAV-VEGF-treated rats, but, surprisingly, capillaries were not. We conclude that intra-arterial AAV-mediated gene transfer of AAV-VEGF(165) normalizes muscle oxygen tension and leads to arteriogenesis in rats with severe hindlimb ischemia.

Overexpression of endothelial nitric oxide synthase increases skeletal muscle blood flow and oxygenation in severe rat hind limb ischemia

OBJECTIVE Although nitric oxide (NO) has a critical role in angiogenesis, the therapeutic potential of NO synthase overexpression in severe ischemia remains undefined. We tested the hypothesis that overexpression of endothelial NO synthase (eNOS) would improve tissue perfusion in severe hind limb ischemia. METHODS Severe hind limb ischemia was induced in 122 adult male Sprague-Dawley rats. Ten days after the induction of hind limb ischemia, vascular isolation and intraarterial delivery of an adenoviral vector encoding eNOS (AdeNOS), a control adenoviral vector (AdE1), or phosphate-buffered saline solution (PBS) was performed. Skeletal muscle blood flow, muscle oxygen tension, angiography, and immunohistochemistry for capillary counts were measured. RESULTS Gene transfer of AdeNOS increased eNOS protein expression and enzyme activity. Two weeks after gene transfer, skeletal muscle blood flow was fourfold higher in eNOS-transduced than in AdE1-transduced or PBS treated rats and was similar to exercise-induced maximal flow in nonischemic muscle. eNOS overexpression increased muscle oxygen tension in a titer-dependent fashion. This increase persisted 1 month after transduction, even though eNOS enzyme activity had declined to normal levels. Angiography and capillary counts showed that eNOS overexpression increased the size and number of collateral arteries, but did not significantly increase the capillary-muscle fiber ratio. CONCLUSIONS eNOS overexpression in an ischemic rat hind limb significantly increased skeletal muscle blood flow, muscle oxygen tension, and collateral arteries (arteriogenesis). Furthermore, eNOS overexpression did not result in capillary angiogenesis above control levels. These studies demonstrate the potential for eNOS overexpression as treatment for severe limb ischemia in human beings.

Effects of retroviral-mediated tissue plasminogen activator gene transfer and expression on adherence and proliferation of canine endothelial cells seeded onto expanded polytetrafluoroethylene

PURPOSE Seeding prosthetic arterial grafts with genetically modified endothelial cells (ECs) has the potential to substantially improve graft function. However, preliminary applications suggest that grafts seeded with retrovirally transduced ECs yield a significantly lower percent surface coverage than those seeded with nontransduced ECs. The objective of this study was to test the hypothesis that canine ECs transduced with the human tissue plasminogen activator (tPA) gene would have a lower rate of adherence to pretreated expanded polytetrafluoroethylene (ePTFE) both in vitro and in vivo and that they would proliferate at a slower rate on pretreated ePTFE in vitro. METHODS Early passage ECs derived from canine external jugular vein were transduced with the retroviral MFG vector containing the gene for human tPA. ECs exposed to media alone served as controls. Iodine 125-labeled ECs were seeded in vitro onto ePTFE graft segments pretreated with canine whole blood, fibronectin (50 micrograms/ml), or media alone, and the percent of ECs adherent at 1 hour were determined (n = 3). Additional tPA-transduced and -nontransduced ECs were grown for 10 days on either fibronectin (50 micrograms/ml)-pretreated ePTFE wafers or tissue culture plastic pretreated with gelatin (1%) or fibronectin (50 micrograms/ml), and the EC proliferation rates were determined (n = 3). Furthermore, 125I-labeled ECs were seeded onto fibronectin (50 micrograms/ml)-pretreated ePTFE graft segments implanted as carotid and femoral artery interposition grafts (n = 3). The grafts were harvested after 1 hour, and the percent of ECs adherent was determined. RESULTS Human tPA was detected by immunohistochemical staining in 61% +/- 5% of the transduced ECs and was expressed at 35.4 +/- 12.9 ng/hr/10(6) cells. Fibronectin and whole blood pretreatment of the ePTFE grafts led to greater EC adherence in vitro than did media alone (90.9% +/- 5.3% vs 77.8% +/- 5.8% vs 4.7% +/- 1.1%, p < or = 0.05). No significant difference in the rates of adherence or proliferation was seen in vitro between the transduced and nontransduced ECs. No significant difference in proliferation was found for the transduced ECs on the three matrices tested in vitro. In contrast, adherence of the transduced ECs in vivo was significantly lower than that of nontransduced ECs (64.7% +/- 2.1% vs 73.7% +/- 4.1%, p < or = 0.05) 1 hour after implantation. CONCLUSIONS Lower rates of surface endothelialization by genetically modified ECs in vivo do not appear to be due to an impaired capacity to initially adhere or proliferate on the synthetic graft but may result from decreased adherence after exposure to in vivo hemodynamic forces.