Molecular characterization of post-thrombotic syndrome

The post-thrombotic syndrome represents a poorly understood and significant vascular health problem. This review focuses on our current understanding of the pathogenesis of post-thrombotic syndrome. We emphasize the cellular and molecular mechanisms that are responsible for the critical components of post-thrombotic syndrome. These include the initiation of deep venous thrombosis, the pathogenesis of elevated venous pressure, and the factors responsible for nonhealing of venous stasis ulcers

Differential transcriptional activation of matrix metalloproteinase-2 and membrane type-1 matrix metalloproteinase by experimental deep venous thrombosis and thrombin

ObjectiveResolution of deep venous thrombosis (DVT) is involved in the pathogenesis of postthrombotic syndrome. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that are critical in angiogenesis and tissue remodeling. We hypothesized that MMP-2 and its membrane-bound activator membrane type-1 matrix metalloproteinase (MT1-MMP) expression would be expressed and activated during the resolution of DVT.MethodsDVT was generated by caval ligation in wild-type and MMP-2 transgenic reporter mice. Ligated and sham-operated (control) cavae were analyzed for MMP-2 transcription (β–galactosidase activity in MMP-2 reporter mice) and MT1-MMP mRNA by real-time polymerase chain reaction. MMP-2 activity was determined by zymography, and immunohistochemical staining for β–galactosidase and MT1-MMP protein was used to localize expression. Human umbilical vascular endothelial cells (HUVEC) were treated with 10 U/mL thrombin and MMP-2 and MT1-MMP mRNA levels and MMP-2 activity was determined.ResultsMMP-2 activity increased 71% (n = 5, P < .05) at day 8 in ligated vs control cavae by zymography. β-galactosidase activity showed a 1.2-fold (n = 8, P < .05) and 1.7-fold (n = 8, P < .05) induction in MMP-2 transcription at day 3 and day 8, respectively. No significant MT1-MMP gene induction was seen at day 3 in ligated vs control cavae, but MT1-MMP mRNA was upregulated 2.5-fold (n = 8, P < .05) in ligated cavae at day 8. Immunohistochemical staining localized MMP-2 and MT1-MMP expression to the vein wall and cellular infiltrates of the thrombus. Thrombin-treated HUVEC showed differential responses of MMP-2 and MT1-MMP. Zymography of conditioned media and cell lysates illustrated a 220% (152.6 ± 8.6 vs 69.445 ± 5.46 pixels/unit area, n = 5, P < .05) and 150% (74.1 ± 7.3 vs 49.2 ± 5.7 pixels/unit area, n = 5, P < .05) increases in MMP-2 activity respectively. MMP-2 mRNA levels were downregulated 30% (0.48 ± 0.023 vs 0.63 ± 0.035 copies of MMP-2 mRNA/copy GAPDH, n = 5, P < .05), whereas MT1-MMP message was upregulated 250% (0.147 ± 0.009 vs 0.059 ± 0.005 copies of MT1-MMP mRNA/copy GAPDH, n = 5, P < .05).ConclusionsResolution of DVT is associated with increased MMP-2 transcription and activity as well as MT1-MMP expression. Thrombin may mediate the increase in MT1-MMP noted in DVT. This is the first article studying MMP-2 and MT1-MMP transcription in DVT. These findings add DVT resolution to the class of inflammatory and fibrotic disorders in which transcriptional activation of the MT1-MMP/MMP-2 genes occurs and identify a potential therapeutic target to modulate this clinically relevant process.Clinical RelevancePostthrombotic syndrome remains a significant clinical problem after deep venous thrombosis (DVT), but the cellular and molecular mechanisms involved in thrombus resolution and vein wall fibrosis remain undefined. Matrix metalloproteinase (MMP) enzymes are critical to cell migration and matrix breakdown. We identify gene transcription and activity of two MMP isoforms, MMP-2 and MMP-14 (membrane type MMP 1, MT1-MMP) in the resolution phase of experimental DVT and in thrombin-treated endothelial cells. These studies define new proteases potentially important to resolution of DVT and development of postthrombotic syndrome

Effect of adenoviral titer and instillation pressure on gene transfer efficiency to arterial and venous grafts ex-vivo

AbstractObjective: Adenoviral-mediated gene transfer to arterial and venous grafts has potential in the treatment of a number of vascular diseases. Despite widespread use of these vectors to mediate gene transfer to blood vessel walls, the optimal transduction conditions for each type of vessel has yet to be determined. Our objective was to study the effect of adenoviral titer and instillation pressure on efficiency of gene transfer to arterial and venous grafts ex-vivo. Methods: Jugular vein and carotid artery segments of 8 cm were harvested from Yorkshire Cross pigs. Tissue culture media or different titers of an adenoviral vector encoding human placental alkaline phosphatase (hpAP) were instilled into venous and arterial grafts at 0 mm Hg or 80 to 100 mm Hg of pressure and bathed externally in the same solution at 37° C for 30 minutes. The grafts were rinsed, opened longitudinally, and incubated in culture media at 37° C for 48 hours. Grafts were fixed and stained for hpAP transgene expression to quantitate percent luminal transduction or homogenized for alkaline phosphatase (AP) activity to determine total transmural transduction. Results: For venous grafts, the percent luminal area stained for hpAP was greatest with 108 plaque-forming units/mL at 0 mm Hg (81% ± 7%) and decreased with increasing titers (53% ± 9% at 109 pfu/mL and 44% ± 11% at 5 × 109 pfu/mL; n = 7; P < .05). No increase in percent luminal area stain was achieved with an instillation pressure of 80 to 100 mm Hg at any viral titer. The inverse finding was observed in arterial grafts. For arterial grafts, the greatest percent luminal area stained was achieved with 5 × 109 pfu/mL at 80 to 100 mm Hg (76% ± 7%). An instillation pressure of 80 to 100 mm Hg increased the percent luminal area stained at 108 pfu/mL from 31% ± 9% to 66% ± 8% (n = 8; P = .01). For venous grafts, total AP activity peaked with 109 pfu/mL at 0 mm Hg and decreased with an instillation pressure of 80 to 100 mm Hg (30.6 ± 9.7 U/mg versus 10.9 ± 2.5 U/mg; n = 7; P < .01). However, for arterial grafts, total AP activity peaked with 5 × 109 pfu/mL (0 mm Hg) and increased with an instillation pressure of 80 to 100 mm Hg (32.8 ± 9.9 U/mg versus 63.4 ± 20.5 U/mg; n = 8; P < .05). Conclusion: High transduction efficiency can be achieved with adenoviral-mediated gene transfer of arterial and venous grafts. Gene transfer with the vascular graft's physiologic pressure conditions improved transduction efficiency for the artery (80 to 100 mm Hg) and vein (0 mm Hg). Comprehensive analysis of adenoviral transduction conditions is important to realize the full promise of adenoviral-mediated gene transfer. (J Vasc Surg 2002;36:263-70.

The effect of gradual or acute arterial occlusion on skeletal muscle blood flow, arteriogenesis, and inflammation in rat hindlimb ischemia

BackgroundCurrent experimental models of critical limb ischemia are based on acute ischemia rather than on chronic ischemia. Human peripheral vascular disease is largely a result of chromic ischemia. We hypothesized that a model of chronic hindlimb ischemia would develop more collateral arteries, more blood flow, and less necrosis and inflammation than would acute hindlimb ischemia. We therefore developed a rat model of chronic hindlimb ischemia and compared the effects of chronic ischemia with those of acute ischemia on hindlimb skeletal muscle.MethodsAcute or chronic ischemia was induced in 36 male Sprague-Dawley rats. Chronic ischemia caused blood flow, as measured by laser Doppler scanning and confirmed by muscle oxygen tension measurements, to gradually decrease over 1 to 2 weeks after operation.ResultsHistologic analysis showed chronic hindlimb ischemia better preserved muscle mass and architecture and stimulated capillary angiogenesis, while lacking the muscle necrosis and inflammatory cell infiltrate seen after acute ischemia. Surprisingly, the chronic ischemia group recovered dermal blood flow more slowly and less completely than did the acute ischemia group, as measured by laser Doppler (0.66 ± 0.02 vs 0.76 ± 0.04, P < .05) and tissue oxygen tension (0.61 ± 0.06 vs 0.81 ± 0.05, P < .05) at 40 days postoperatively. Consistent with poorer blood flow recovery, chronic ischemia resulted in smaller diameter collateral arteries (average diameter of the five largest collaterals on angiogram was 0.01 ± 0.0003 mm vs 0.013 ± 0.0007 mm for acute, P < .005 at 40 days postoperatively). Acute ischemia resulted in decreased tissue concentrations of vascular endothelial growth factor (VEGF) (0.96 ± 0.23 pg/mg of muscle for acute vs 4.4 ± 0.75 and 4.8 ± 0.75 pg/mg of muscle for unoperated and chronic, respectively, P < .05 acute vs unoperated), and in increased tissue concentrations of interleukin (IL)-1β (7.3 ± 4.0 pg/mg of muscle for acute vs undetectable and 1.7 ± 1.6pg/mg of muscle for unoperated and chronic, respectively, P < 0.05 acute vs unoperated).ConclusionsWe describe here the first model of chronic hindlimb ischemia in the rat. Restoration of blood flow after induction of hindlimb ischemia is dependent on the rate of arterial occlusion. This difference in blood flow recovery correlates with distinct patterns of muscle necrosis, inflammatory cell infiltration, and cytokine induction in the ischemic muscle. Differences between models of acute and chronic hindlimb ischemia may have important consequences for future studies of mechanisms regulating arteriogenesis and for therapeutic approaches aimed at promoting arteriogenesis in humans suffering from critical limb ischemia.Clinical relevanceDespite the substantial clinical differences between acute and chronic ischemia, researchers attempting to develop molecular therapies to treat critical limb ischemia have only tested those therapies in experimental models of acute hindlimb ischemia. We present here a novel model of chronic hindlimb ischemia in the rat. We further demonstrate that when hindlimb ischemia is developed chronically, collateral artery development is poorer than when hindlimb ischemia is developed acutely. These findings suggest that further tests of molecular therapies for critical limb ischemia should be performed in chronic hindlimb ischemia models rather than in acute hindlimb ischemia models

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

AbstractObjectiveAlthough 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.MethodsSevere 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.ResultsGene 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.ConclusionseNOS 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

SS25. Cryopreserved Venous Allograft: An Alternative Conduit for Reconstruction of Infected Prosthetic Aortic Grafts

Olivé Milián, ArmandPla general picat del mosaic format per tres cercles: el central, amb un sol de color ocre sobre un cel blau. El sol, somrient, està encarat cap al sud. Aquest està envoltat d'un primer anell dentat i un segon on s'hi representen les fases d

Mechanisms of inhibition of vascular smooth muscle cell function by nitric oxide.

Nitric oxide (NO), an important biologic regulator of vascular function, is known to inhibit the growth of cultured vascular smooth muscle cells. The anti-proliferative mechanisms and cell cycle effects of nitric oxide remain undefined. This thesis tested the hypothesis that nitric oxide inhibits smooth muscle proliferation by inhibiting progression at a specific point in the cell cycle. The inhibitory effect of exogenous nitric oxide occurred during S phase in synchronized rat aortic smooth muscle cells. This S phase inhibition occurred after the restriction point of commitment to cell proliferation late in G1 phase, which is distinct from the point of inhibition of other endogenous inhibitors of proliferation. This S phase effect is not mimicked by cGMP, a second messenger for NO. The mechanism of S phase inhibition was partially reversed by bypass of ribonucleotide reductase, suggesting that inhibition by NO occurs at this point. In cycling cells NO caused immediate inhibition of DNA synthesis, confirming the effects seen in synchronized cells. Prolonged treatment with NO did not synchronize cells at the G1/S border, as hydroxyurea did, but instead induced a G0 state characterized by a prolonged delay before DNA synthesis resumed after removal of NO. This G0 state was not due to inhibition of either protein synthesis or mitochondrial respiration. NO donors also inhibited migration of smooth muscle cells in culture independent of inhibition of proliferation. This inhibition of migration was mimicked by an exogenous cGMP analog, suggesting the involvement of guanylate cyclase in mediating this effect. Inhibition of smooth muscle cell migration may be another mechanism by which NO inhibits neointimal hyperplasia in vivo. DNA synthesis and proliferation of cultured endothelial cells was also inhibited by NO donors at concentrations which inhibit smooth muscle proliferation. This may be one mechanism by which pathologic production of NO in diseased vessels inhibits re-endothelialization. NO is the first endogenous substance which inhibits cell growth in S phase, a process that may be important in regulation of proliferation of cells that have lost normal G1 mechanisms of growth control.Ph.D.PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104700/1/9542951.pdfDescription of 9542951.pdf : Restricted to UM users only