Carotid Injury Research Articles

Novel use of a Sengstaken–Blakemore tube during a neck exploration of a carotid injury: A case report

Penetrating neck trauma can injure the major blood vessels, airway, gastrointestinal system, and neurological system. We present a case where a Sengstaken–Blakemore tube was emergently placed during surgical exploration of a stab wound to the neck to tamponade bleeding until surgical control was obtained and the vascular injuries were managed.

Activation of transient receptor potential vanilloid 1 accelerates re-endothelialization and inhibits neointimal formation after vascular injury

Transient receptor potential vanilloid 1 (TRPV1) is an important regulator of endothelial function, but the effects of TRPV1 on endothelial recovery and neointimal formation after vascular injury remain elusive. We tested the effects of activating TRPV1 using capsaicin on re-endothelialization and neointimal formation after wire-induced injury of the carotid artery in mice. The human umbilical vein endothelial cells (HUVECs) were treated with the TRPV1 agonist capsaicin, its antagonist capsazepine, intracellular calcium chelator BAPTA, or mitofusin 2 (Mfn2)-specific short interfering RNA (siRNA). The migration, proliferation, mitochondrial morphology, membrane potential, and adenosine triphosphate production were measured. The carotid artery wire injury procedure was performed in male TRPV1 knockout mice and C57BL/6J wild-type (WT) mice that were then treated with or without Mfn2 siRNA. The re-endothelialization and neointimal formation were evaluated. Capsaicin significantly enhanced the migration and proliferation of HUVECs. Both capsazepine and BAPTA abolished capsaicin-induced migration and proliferation of HUVECs. In addition, capsaicin stimulated the formation of reticular mitochondria, augmented mitochondrial membrane potential, increased adenosine triphosphate production, and upregulated Mfn2. However, these effects were attenuated by knockdown of Mfn2 with specific siRNA. Dietary capsaicin markedly accelerated re-endothelialization and inhibited neointimal formation in WT mice but not in TRPV1 knockout mice. Moreover, Mfn2 siRNA also attenuated capsaicin-induced enhancement of endothelial recovery and suppression of neointimal hyperplasia in WT mice. Activation of TRPV1 with capsaicin attenuates neointimal formation by accelerating re-endothelialization through upregulation of Mfn2.

Platelet Pitp-Alpha Promotes Thrombin Generation and the Dissemination of Tumor Metastasis, but Has Minimal Effect on Vascular Plug Formation

Phosphatidylinositol (PtdIns) is a relatively rare phospholipid in cell membranes. However, because of its unique ability to be transiently phosphorylated, it is critical for platelet signaling and vascular plug formation. Phosphatidylinositol transfer proteins (PITPs) facilitate the transfer of aqueous insoluble phosphatidylinositol in vitro from one cell membrane to another. Although murine platelets contain multiple PITP variants, PITPα is by far the most abundant isoform. Platelets are best known for their contribution to hemostasis, however, several lines of evidence indicate that they also contribute to tumor metastasis formation. To test the hypothesis that platelet phosphoinositide signaling contributes to tumor dissemination, we asked whether deletion of platelet PITPα impacts tumor metastasis formation by engineering PITPαfl/fl PF4Cre+ mice that lack PITPα only within their platelets and megakaryocytes. We found that loss of PITPα in platelets decreased PtdIns(4)P and PtdIns(4,5)P2 production by 30-40%. Even more striking was the 80% reduction in IP3 formation following thrombin-stimulation. However, we found no significant defect in the platelet ex vivo aggregation in response to typical doses of most of the platelet agonists, and only a small defect in response to low doses of thrombin. We investigated whether platelets lacking PITPα normally formed thrombi in vivo by using three well-established murine models. First, we observed an extremely minor (but statistically real) prolongation of the tail bleeding time in the PITPαfl/fl PF4Cre+ mice. Second, we found that mice lacking PITPα in their platelets had no defect in forming intravascular clots in response to a chemical induced carotid injury. Finally, we observed that thrombosis and platelet a-granule secretion in response to a laser-induced injury were completely normal in the knockout mice. Therefore, despite the biochemical defect in phosphoinositide signaling induced by the loss of PITPα, there was essentially no hemostastic defect. To determine whether PITPα-mediated phosphoinositide metabolism in platelets is required for tumor dissemination, we utilized a well-characterized B16F10 melanoma model of tumor metastasis. We observed that lung metastasis formation was reduced by 47%±18% in mice lacking PITPα in their platelets. We also found that during the first 3 hours after tumor injection, the control mice, but not the PITPαfl/fl PF4Cre+ mice, developed a rapid and transient thrombocytopenia. Histology analysis of the lung tissue at this time point revealed the presence of 30% more clots in the lung tissue of control mice. Further analysis showed that these thrombi were actually heterogenous complexes composed of tumor cells surrounded by platelets and fibrin. To understand why platelet PITPα influences tumor-induced fibrin formation, we investigated the ability of tumor cells to cause thrombin generation in platelet rich plasma derived from PITPαfl/fl PF4Cre+ and PITPαfl/fl PF4Cre- mice. We observed that the loss of PITPα in platelets resulted in an 88% reduction of thrombin generation compared to the controls. Furthermore, we found that PITPαfl/fl PF4Cre+ platelets also have impaired Annexin V binding suggesting that the defective fibrin formation seen in PITPαfl/fl PF4Cre+ mice is likely due to a role of PITPα in the exposure of phosphatidylserine on the platelet surface that is required for thrombin generation. Finally, we observed a mucosal immune response composed of NK-cells, T-cells, and neutrophils that was strikingly hyperplastic in mice lacking platelet PITPα at 48 hours after tumor injection, but was essentially absent in the control mice. Together, these findings demonstrate that PITPα-mediated phosphoinositide metabolism within platelets is not essential for platelet plug formation in vivo, but is required for the dissemination of tumors in vivo. Our work further demonstrates that platelet PITPα is required for tumor-induced phosphatidylserine exposure and thrombin generation. This process induces a shroud of platelets and fibrin that surround the surface of tumor cells, and thereby protects the tumors from elimination by the mucosal immune system. These results demonstrate that it is possible to clearly distinguish the platelet signaling processes required for platelet plug formation from those processes that augment metastasis formation. DisclosuresNo relevant conflicts of interest to declare.

Chronic, not acute, skin-specific inflammation promotes thrombosis in psoriasis murine models.

BackgroundPsoriasis patients exhibit an increased risk of atherothrombotic events, including myocardial infarction and stroke. Clinical evidence suggests that psoriasis patients with early onset and more severe disease have the highest risk for these co-morbidities, perhaps due to the extent of body surface involvement, subsequent levels of systemic inflammation, or chronicity of disease. We sought to determine whether acute or chronic skin-specific inflammation was sufficient to promote thrombosis.MethodsWe used two experimental mouse models of skin-specific inflammation generated in either an acute (topical Aldara application onto wild-type C57Bl/6 mice for 5 days) or chronic (a genetically engineered K5-IL-17C mouse model of psoriasiform skin inflammation) manner. Arterial thrombosis was induced using carotid artery photochemical injury (Rose Bengal-green light laser) and carotid artery diameters were measured post-clot formation. We also examined measures of clot formation including prothrombin (PT) and activated partial thromboplastin time (aPTT). Skin inflammation was examined histologically and we profiled plasma-derived lipids. The number of skin-draining lymph-node (SDLN) and splenic derived CD11b+Ly6Chigh pro-inflammatory monocytes and CD11b+Ly6G+ neutrophils was quantified using multi-color flow cytometry.ResultsMice treated with topical Aldara for 5 days had similar carotid artery thrombotic occlusion times to mice treated with vehicle cream (32.2 ± 3.0 vs. 31.4 ± 2.5 min, p = 0.97); in contrast, K5-IL-17C mice had accelerated occlusion times compared to littermate controls (15.7 ± 2.1 vs. 26.5 ± 3.5 min, p < 0.01) while carotid artery diameters were similar between all mice. Acanthosis, a surrogate measure of inflammation, was increased in both Aldara-treated and K5-IL-17C mice compared to their respective controls. Monocytosis, defined as elevated SDLN and/or splenic CD11b+Ly6Chigh cells, was significantly increased in both Aldara-treated (SDLN: 3.8-fold, p = 0.02; spleen: 2.0-fold, p < 0.01) and K5-IL-17C (SDLN: 3.4-fold, p = 0.02; spleen: 3.5-fold, p < 0.01) animals compared to controls while neutrophilia, defined as elevated SDLN and/or splenic CD11b+Ly6G+ cells, was significantly increased in only the chronic K5-IL-17C model (SDLN: 11.6-fold, p = 0.02; spleen: 11.3-fold, p < 0.01). Plasma-derived lipid levels, PT and aPTT times showed no difference between the Aldara-treated mice or the K5-IL-17C mice and their respective controls.ConclusionsChronic, but not acute, skin-specific inflammation was associated with faster arterial thrombotic occlusion. Increased numbers of splenic and SDLN monocytes were observed in both acute and chronic skin-specific inflammation, however, increased splenic and SDLN neutrophils were observed only in the chronic skin-specific inflammation model. Understanding the cellular response to skin-specific inflammation may provide insights into the cellular participants mediating the pathophysiology of major adverse cardiovascular events associated with psoriasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0738-z) contains supplementary material, which is available to authorized users.

The Natural History and Management of Petrous Apex Cholesterol Granulomas.

To evaluate the clinical and radiographic characteristics of cholesterol granulomas of the petrous apex, as well as the outcomes of operative and conservative management. Retrospective chart review. Two independent tertiary academic referral centers. Adult and pediatric patients with cholesterol granulomas of the petrous apex were identified from the experience of two separate centers. Patients were included after radiographic diagnosis and clinical evaluation. All patients with less than 6 months of follow-up and those with iatrogenic postoperative cholesterol granulomas were excluded. Demographic information, presenting symptoms, imaging characteristics, treatment strategies, and outcomes were recorded. Patients were evaluated on the basis of symptom and radiographic evolution during time, with or without operative intervention. Ninety petrous apex cholesterol granulomas were analyzed (57.8% females, 55.6% right-sided). The average age at presentation was 43.1 years (median 42.0, range 8.0-77.0 years). The most common presenting symptom was headache (56.7%), and the average lesion size in the greatest dimension was 2.1 cm (median 1.7, range 0.7-5.0 cm). During a mean follow-up of 46.0 months, no cases of spontaneous rupture or carotid injury occurred. Twenty-three patients (25.6%) ultimately underwent surgical management, most commonly for intractable headache, and only 47.8% of these patients experienced durable symptom improvement by their last postoperative follow-up. Many cholesterol granulomas of the petrous apex remain stable during time and can be safely managed with primary observation. Surgery should be reserved for lesions that are causing, or threatening, neurologic dysfunction because of mass effect or erosion of critical structures such as the otic capsule. Cranial neuropathy associated with cholesterol granuloma may improve after operative management; however, symptoms such as headache and dizziness are less likely to benefit from surgery. As a general guideline, in the presence of a radiologically stable cholesterol granuloma in the petrous apex, alternative etiologies for headache and dizziness should be considered and treated before offering surgical intervention.

Abstract 16419: Epigenetic Readers: An Attractive Way to Tackle PAH and the Associated Vascular Comorbidities

Introduction: Pulmonary arterial hypertension (PAH) is a devastating vasculopathy. Recent progress in understanding its pathogenesis led to an increase in patients’ quality of life. With ageing, PAH patients now develop comorbidities including coronary artery diseases (CAD), further challenging their therapeutic management. We previously reported that CAD and PAH share many similarities, including vascular smooth muscle cells (VSMCs) pro-proliferative/anti-apoptotic and metabolic abnormalities. We could speculate that PAH and CAD development share many pathophysiological pathways. In cancer, the epigenetic reader Bromodomain-containing protein 4 (BRD4) increases transcription of many oncogenes contributing to the proliferative/apoptosis imbalance. Interestingly, BRD4 was shown to interact with miR-204 and NFAT, both critical players in PAH and CAD. We hypothesized that BRD4 is a central actor of VSMCs impairment contributing to vascular remodeling observed in PAH and CAD. Method/Results: We demonstrated that remodeled distal pulmonary arteries from PAH patients exhibit a miR-204-dependent increase in BRD4 expression compared to controls (qRT-PCR, western blot, n=10-15, p&lt;0.01). This phenotype is also observed in VSMC of remodeled coronary arteries from both PAH patients with CAD and non-PAH CAD patients compared to non-CAD patients (n=10, p&lt;0.05). In vitro, we showed that BRD4 increases NFAT, Bcl-2 and Survivin (n=6, p&lt;0.01) expression, triggering both proliferation (Ki67, p&lt;0.05) and resistance to apoptosis (AnnexinV, p&lt;0.05) while decreasing spare respiratory capacity (Seahorse XF, n=4, p&lt;0.05) and hyperpolarizing mitochondrial membranes (TMRM, p&lt;0.05). All these abnormalities were reversed upon BRD4 inhibition (JQ1 or siBRD4). In vivo, JQ1/siBRD4 reverses established PAH and prevents carotid stenosis in Sugen/hypoxia and carotid injury rat models respectively (n=8-10/group). Conclusion: Comorbidities in PAH such as CAD has become a major clinical problem. Thus, targeting mechanisms implicated in both PAH and subsequent comorbidities is warranted. To this aim, we propose that BRD4 might represent an attractive target and since BRD4 inhibitors are presently in clinical trials further potentiates its uses in PAH.

Endothelial Krüppel-Like Factor 4 Mediates the Protective Effect of Statins against Ischemic AKI.

Endothelial cells participate in the pathophysiology of ischemic AKI by increasing the expression of cell adhesion molecules and by recruiting inflammatory cells. We previously showed that endothelial Krüppel-like factor 4 (Klf4) regulates vascular cell adhesion molecule 1 (Vcam1) expression and neointimal formation after carotid injury. In this study, we determined whether endothelial Klf4 is involved in ischemic AKI using endothelial Klf4 conditional knockout (Klf4 cKO) mice generated by breeding Tek-Cre mice and Klf4 floxed mice. Klf4 cKO mice were phenotypically normal before surgery. However, after renal ischemia-reperfusion injury, Klf4 cKO mice exhibited elevated serum levels of urea nitrogen and creatinine and aggravated renal histology compared with those of Klf4 floxed controls. Moreover, Klf4 cKO mice exhibited enhanced accumulation of neutrophils and lymphocytes and elevated expression of cell adhesion molecules, including Vcam1 and Icam1, in injured kidneys. Notably, statins ameliorated renal ischemia-reperfusion injury in control mice but not in Klf4 cKO mice. Mechanistic analyses in cultured endothelial cells revealed that statins increased KLF4 expression and that KLF4 mediated the suppressive effect of statins on TNF-α-induced VCAM1 expression by reducing NF-κB binding to the VCAM1 promoter. These results provide evidence that endothelial Klf4 is renoprotective and mediates statin-induced protection against ischemic AKI by regulating the expression of cell adhesion molecules and concomitant recruitment of inflammatory cells.

Impaired LRP6-TCF7L2 Activity Enhances Smooth Muscle Cell Plasticity and Causes Coronary Artery Disease.

Mutations in Wnt-signaling coreceptor LRP6 have been linked to coronary artery disease (CAD) by unknown mechanisms. Here, we show that reduced LRP6 activity in LRP6(R611C) mice promotes loss of vascular smooth muscle cell (VSMC) differentiation, leading to aortic medial hyperplasia. Carotid injury augmented these effects and led to partial to total vascular obstruction. LRP6(R611C) mice on high-fat diet displayed dramatic obstructive CAD and exhibited an accelerated atherosclerotic burden on LDLR knockout background. Mechanistically, impaired LRP6 activity leads to enhanced non-canonical Wnt signaling, culminating in diminished TCF7L2 and increased Sp1-dependent activation of PDGF signaling. Wnt3a administration to LRP6(R611C) mice improved LRP6 activity, led to TCF7L2-dependent VSMC differentiation, and rescued post-carotid-injury neointima formation. These findings demonstrate the critical role of intact Wnt signaling in the vessel wall, establish a causal link between impaired LRP6/TCF7L2 activities and arterial disease, and identify Wnt signaling as a therapeutic target against CAD.

Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis

AbstractAutophagy is important for maintaining cellular homeostasis, and thus its deficiency is implicated in a broad spectrum of human diseases. Its role in platelet function has only recently been examined. Our biochemical and imaging studies demonstrate that the core autophagy machinery exists in platelets, and that autophagy is constitutively active in resting platelets. Moreover, autophagy is induced upon platelet activation, as indicated by agonist-induced loss of the autophagy marker LC3II. Additional experiments, using inhibitors of platelet activation, proteases, and lysosomal acidification, as well as platelets from knockout mouse strains, show that agonist-induced LC3II loss is a consequence of platelet signaling cascades and requires proteases, acidic compartments, and membrane fusion. To assess the physiological role of platelet autophagy, we generated a mouse strain with a megakaryocyte- and platelet-specific deletion of Atg7, an enzyme required for LC3II production. Ex vivo analysis of platelets from these mice shows modest defects in aggregation and granule cargo packaging. Although these mice have normal platelet numbers and size distributions, they exhibit a robust bleeding diathesis in the tail-bleeding assay and a prolonged occlusion time in the FeCl3-induced carotid injury model. Our results demonstrate that autophagy occurs in platelets and is important for hemostasis and thrombosis.

The Natural History of Indeterminate Blunt Cerebrovascular Injury.

The Denver criteria grade blunt cerebrovascular injuries (BCVIs) but fail to capture many patients with indeterminate findings on initial imaging. To evaluate outcomes and clinical significance of indeterminate BCVIs (iBCVIs). A retrospective review of all patients treated for BCVIs at our institution from January 1, 2007, through July 31, 2014, was completed. Patients were divided into 2 groups: those with true BCVIs as defined by the Denver criteria and those with iBCVIs, which was any initial imaging suggestive of a cerebrovascular arterial injury not classifiable by the Denver criteria. Primary outcomes were rate of resolution of iBCVIs, freedom from cerebrovascular accident (CVA) or transient ischemic attack (TIA), and 30-day mortality. We identified 100 patients with 138 BCVIs: 79 with true BCVIs and 59 with iBCVIs. With serial imaging, 23 iBCVIs (39.0%) resolved and 21 (35.6%) remained indeterminate, whereas 15 (25.4%) progressed to true BCVI. The rate of CVA or TIA in the iBCVI group was 5.1% compared with 15.2% in the true BCVI group (P = .06). Of the 15 total CVAs or TIAs, 11 (73.3%) resulted from carotid injury and 4 (26.7%) from vertebral artery occlusion (P = .03). By Kaplan-Meier analysis, there was no difference in freedom from CVA or TIA for the 2 groups (P = .07). Median clinical follow-up was 91 days. Overall and 30-day mortality for the entire series were 17.4% and 15.2%, respectively. There was no difference in long-term or 30-day mortality between true BCVI and iBCVI groups. Detection of iBCVI has become a common clinical conundrum with improved and routine imaging. Indeterminate BCVI is not completely benign, with 25.4% demonstrating anatomical progression to true BCVI and 5.1% developing cerebrovascular symptoms. We therefore recommend serial imaging and antiplatelet therapy for iBCVI.

Digoxin inhibits PDGF-BB-induced VSMC proliferation and migration through an increase in ILK signaling and attenuates neointima formation following carotid injury

The increased proliferation and migration of vascular smooth muscle cells (VSMCs) are key events in the development of artery restenosis following percutaneous coronary intervention. Digoxin has long been used in the treatment of heart failure and has been shown to inhibit the proliferation of cancer cells through multiple pathways. However, the potential role of digoxin in the regulation of VSMC proliferation and migration and its effectiveness in the treatment of cardiovascular diseases, such as restenosis, remains unexplored. In the present study, we demonstrate that digoxin-induced growth inhibition is associated with the downregulation of CDK activation and the restoration of p27Kip1 levels in platelet-derived growth factor (PDGF)-stimulated VSMCs. In addition, we found that digoxin restored the PDGF-BB-induced inhibition of integrin linked kinase (ILK) expression and prevented the PDGF-BB-induced activation of glycogen synthase kinase (GSK)-3β. Furthermore, digoxin inhibited adhesion molecule and extracellular matrix relative protein expression. Finally, we found that digoxin significantly inhibited neointima formation, accompanied by a decrease in cell proliferation following vascular injury in rats. These effects of digoxin were shown to be mediated, at least in part, through an increase in ILK/Akt signaling and a decrease in GSK-3β signaling in PDGF-BB-stimulated VSMCs. In conclusion, our data demonstrate that digoxin exerts an inhibitory effect on the PDGF-BB-induced proliferation, migration and phenotypic modulation of VSMCs, and prevents neointima formation in rats. These observations indicate the potential therapeutic application of digoxin in the treatment of cardiovascular diseases, such as restenosis.

C is for carotid injury

C is for carotid injury

Mesenchymal Stem Cells Stabilize Atherosclerotic Vulnerable Plaque by Anti-Inflammatory Properties.

Background and objectivesFormation and progression of atherosclerotic vulnerable plaque (VP) is the primary cause of many cardio-cerebrovascular diseases such as acute coronary syndrome and stroke. It has been reported that bone marrow mesenchymal stem cells (MSC) exhibit protective effects against many kinds of diseases including myocardial infarction. Here, we examined the effects of intravenous MSC infusion on a VP model and provide novel evidence of its influence as a therapy in this animal disease model.Subjects and methodsThirty healthy male New Zealand white rabbits were randomly divided into a MSC, VP or stable plaque (SP) group (n = 10/group) and received high fat diet and cold-induced common carotid artery intimal injury with liquid nitrogen to form atherosclerotic plaques. Serum hs-CRP, TNF-α, IL-6 and IL-10 levels were measured by ELISA at 1, 2, 3, 7, 14, 21 and 28 days after MSC transplantation. The animals were sacrificed at 4 weeks after MSC transplantation. Lesions in the right common carotid were observed using H&E and Masson staining, and the fibrous cap/lipid core ratio of atherosclerotic plaques were calculated. The expression of nuclear factor κB (NF-κB) and matrix metalloproteinase 1, 2, 9 (MMP-1,2,9) in the plaque were detected using immunohistochemistry, and apoptotic cells in the plaques were detected by TUNEL. In addition, the level of TNF-α stimulated gene/protein 6 (TSG-6) mRNA and protein were measured by quantitative Real-Time PCR and Western blotting, respectively.ResultsTwo rabbits in the VP group died of lung infection and cerebral infarction respectively at 1 week after plaque injury by liquid nitrogen. Both H&E and Masson staining revealed that the plaques from the SP and MSC groups had more stable morphological structure and a larger fibrous cap/lipid core ratio than the VP group. Serum hs-CRP, TNF-α and IL-6 were significantly down-regulated, whereas IL-10 was significantly up-regulated in the MSC group compared with the VP group. .Immunohistochemistry analysis revealed that NF-κB and MMP expression was reduced in the MSC and SP groups compared to the VP group. Cell apoptosis decreased significantly in both the MSC and SP groups in comparison to the VP group. TSG-6 mRNA and protein expression were higher in the plaques of the MSC group compared to the VP and SP groups.ConclusionsOur study results suggest that MSC transplantation can effectively stabilize vulnerable plaques in atherosclerotic rabbits. This may potentially offer a new clinical application of MSC in atherosclerosis.

Carbon Monoxide Releasing Molecule Accelerates Reendothelialization after Carotid Artery Balloon Injury in Rat

ObjectiveThis study was aimed to investigate the effects of carbon monoxide releasing molecule (CORM-2), a novel carbon monoxide carrier, on the reendothelialization of carotid artery in rat endothelial denudation model. MethodsMale rats subjected to carotid artery balloon injury were treated with CORM-2, inactive CORM-2 (iCORM-2) or dimethyl sulfoxide (DMSO). The reendothelialization capacity was evaluated by Evans Blue dye and the immunostaining with anti-CD31 antibody. The number of circulating endothelial progenitor cells (EPCs) was detected by flow cytometry. The proliferation, migration, and adhesion of human umbilical vein endothelial cells (HUVECs) were assessed by using [3H]thymidine, Boyden chamber and human fibronectin respectively. The expressions of protein were detected by using western blot analysis. ResultsCORM-2 remarkably accelerated the re-endothelialization 5 d later and inhibited neointima formation 28 d later. In addition, the number of peripheral EPCs significantly increased in CORM-2-treated rats than that in iCORM-2 or DMSO-treated rats after 5 d later. In vitro experiments, CORM-2 significantly enhanced the proliferation, migration and adhesion of HUVECs. The levels of Akt, eNOS phosphorylation, and NO generation in HUVECs were also much higher in CORM-2 treated group. Blocking of PI3K/Akt/eNOS signaling pathway markedly suppressed the enhanced migration and adhesion of HUVECs induced by CORM-2. ConclusionsCORM-2 could promote endothelial repair, and inhibit neointima formation after carotid artery balloon injury, which might be associated with the function changes of HUVECs regulated by PI3K/Akt/eNOS pathway.

Sphingosine-1-phosphate (S1P) inhibits TNFA-induced MMP-9 expression (TIME) through S1P2-mediated activation of PTEN

S1P regulates numerous functions in vascular smooth muscle cells (VSMC). We have shown that high S1P levels promoted neointima formation after carotid injury, a process that depends on matrix metalloproteinases (MMP). Here, we tested whether S1P affects basal and cytokine-induced MMP-9 activity in rat and mouse VSMC. S1P potently inhibited TIME as seen on gelatin zymography of the secreted enzyme and gene expression by real time PCR, respectively, without any effect on MMP-2. Using S1P receptor antagonists and VSMC isolated from S1P receptor knockout mice we have identified S1P2 as the S1P receptor responsible for TIME inhibition. S1P had no effect on IkBa phosphorylation and NF-kB activation by TNFa but potently inhibited Akt phosphorylation after TNFa that we found to be required for TIME. S1P inhibited PI3K/Akt by activating the Phosphatase and Tensin homolog (PTEN) pathway by engaging the S1P2 receptor as we could show using PI3K inhibitors, siRNA to PTEN and pharmacological PTEN blockade (bpVHO(pic)). The functional consequence of TIME inhibition by S1P was the abrogation of MMP-dependent TNFa-stimulated VSMC migration in Boyden chamber experiments. In vivo, downregulation of all S1P receptors by maintaining high S1P levels through pharmacological S1P lyase inhibition promoted neointima formation after carotid ligation, while the S1P analogue FTY720 had no effect. FTY720 acts on all S1P receptors except S1P2, and S1P2-deficient mice exhibit the same phenotype as S1P lyase-inhibited. Thus lack of MMP-9 inhibition due to downregulated or missing S1P2 may be responsible for increased neointima formation, a process shown to causally depend on MMP-9.

Pentamethylquercetin (PMQ) reduces thrombus formation by inhibiting platelet function.

Flavonoids exert both anti-oxidant and anti-platelet activities in vitro and in vivo. Pentamethylquercetin (PMQ), a polymethoxylated flavone derivative, has been screened for anti-carcinogenic and cardioprotective effects. However, it is unclear whether PMQ has anti-thrombotic effects. In the present study, PMQ (20 mg/kg) significantly inhibited thrombus formation in the collagen- epinephrine- induced acute pulmonary thrombosis mouse model and the ferric chloride-induced carotid injury model. To explore the mechanism, we evaluated the effects of PMQ on platelet function. We found that PMQ inhibited platelet aggregation and granule secretion induced by low dose agonists, including ADP, collagen, thrombin and U46619. Biochemical analysis revealed that PMQ inhibited collagen-, thrombin- and U46619-induced activation of Syk, PLCγ2, Akt, GSK3β and Erk1/2. Therefore, we provide the first report to show that PMQ possesses anti-thrombotic activity in vivo and inhibited platelet function in vitro, suggesting that PMQ may represent a potential therapeutic candidate for the prevention or treatment of thrombotic disorders.

Increase in Vascular Injury of Sodium Overloaded Mice May be Related to Vascular Angiotensin Modulation.

This study aimed to analyzing the effect of chronic sodium overload upon carotid and femoral injury, and its relation to vascular angiotensin modulation. Male C57Bl6 mice were divided in: control (cont), receiving 1% NaCl solution for 2 weeks (salt-2) or 12 weeks (salt-12). Two-weeks before the end of the study, a 2mm catheter was implanted around the left femoral and carotid arteries to induce injury. Blood pressure (BP) and heart rate (HR) were measured at the end of the study by tail plethysmography. Arteries were collected and prepared for histological analysis to determine arterial thickening and perivascular collagen deposition. Angiotensin II and Ang(1-7) were quantified in fresh arteries using the HPLC method. There were no differences in body weight, BP and HR. Intima/media ratio had a similar increase in both injured arteries of cont and salt-2 mice, but a more pronounced increase was observed in salt-12 mice (31.1±6%). On the other hand, sodium overload modified perivascular collagen deposition, increasing thick fibers (cont: 0.5%; salt-2: 3.4%; salt-12: 0.6%) and decreasing thin fibers (cont: 7.4%; salt-2: 0.5%; salt-12: 6.8%) in non-injured arteries. Injured arteries presented similar collagen fiber distribution. Angiotensin quantification showed increased Ang(1-7) in salt treated mice (salt-2: +72%; salt-12: +45%) with a concomitant decrease in Ang II (salt-2: -54%; salt-12: -60%). Vascular injury increased significantly Ang(1-7) in salt-12 mice (+80%), maintaining Ang II reduction similar to that of a non-injured artery. The lack of changes in BP and HR suggests that the structural changes observed may be due to non-hemodynamic mechanisms such as local renin-angiotensin system. Collagen evaluation suggests that sodium overload induces time-related changes in vascular remodeling. The increase of artery injury with concomitant increase in Ang(1-7) in 12-week treated mice shows a direct association between the duration of salt treatment and the magnitude of vascular injury.

Phloretin Inhibits Platelet-derived Growth Factor-BB-induced Rat Aortic Smooth Muscle Cell Proliferation, Migration, and Neointimal Formation After Carotid Injury.

Abnormal vascular smooth muscle cell proliferation and migration are key factors in many cardiovascular diseases. Here, we investigated the effects of phloretin on platelet-derived growth factor homodimer (PDGF-BB)-induced rat aortic smooth muscle cell (RASMC) proliferation, migration, and neointimal formation after carotid injury. Phloretin significantly inhibited the PDGF-BB-stimulated RASMC proliferation in a concentration-dependent manner (10-100 μM). Also, PDGF-BB-stimulated RASMC migration was inhibited by phloretin at 50 μM. Pretreating RASMC with phloretin dose-dependently inhibited PDGF-BB-induced Akt and p38 mitogen-activated protein kinases activation. Furthermore, phloretin increased p27 and decreased cyclin-dependent kinase 2, CDK4 expression, and p-Rb activation in PDGF-BB-stimulated RASMC in a concentration-dependent manner (10-50 μM). PDGF-BB-induced cell adhesion molecules and matrix metalloproteinase-9 expression were blocked by phloretin at 50 μM. Preincubation with phloretin dose-dependently reduced the intracellular reactive oxygen species production. In vivo study showed that phloretin (20 mg/kg) significantly reduced neointimal formation 14 days after carotid injury in rats. Thus, phloretin may have potential as a treatment against atherosclerosis and restenosis after vascular injury.

61. Vascular Smooth Muscle Cell RNA Aptamers for the Treatment of Cardiovascular Disease

Cardiovascular disease (CVD) is the leading cause of mortality in many countries. Many vascular disorders, including in-stent restenosis, arteriosclerosis, vein graft disease, and cardiac allograft arteriopathy are caused by pathological vascular smooth muscle cell (VSMC) remodeling following injury. An ideal therapeutic intervention would target the VSMCs without impairing the injured vessel re-endothelialization. However, current therapies do not selectively prevent pathological VSMC remodeling leading to impaired re-endothelization, late stent thrombosis and death. Thus, there is a clear need for cell-targeted treatment and prevention options of pathological VSMC remodeling.Our group has described the development of VSMC-specific, aptamers for (1) modulating signaling pathways associated with pathological VSMC remodeling and (2) delivering therapeutic molecules to these cells in vivo. Here we demonstrate that one of these aptamers, Vapt14, inhibits protein kinase B (PKB)/Akt activation and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-beta phosphorylation. In a murine model of carotid injury, treatment of vessels with Vapt14 reduces intimal:medial thickness to levels comparable to that of paclitaxel. Importantly, we confirm that Vapt14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells (PBMCs) in vitro. In addition, we confirm delivery of Vapt14 to VSMC in vitro and in vivo with fluorescence microscopy. Studies are being expanded to evaluate aptamer-mediated delivery of therapeutic biomolecules (e.g. small molecules, RNAi modulators) to areas of vascular injury. In summary this work provides an essential foundation for the translation of cell-targeted RNA therapeutics to multiple hyperplastic vascular diseases.

Abstract 188: Selective Inhibition of Vascular Smooth Muscle Cell Migration by Targeting Plasminogen Activator Inhibitor-1

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of mammalian plasminogen activators and an important regulator of cell migration. We have shown that tiplaxtinin, a small molecule, specific inhibitor of PAI-1, inhibits intimal hyperplasia in a murine vein graft model. However, little is known about the effects of pharmacological inhibition of PAI-1 on vascular cell migration under physiologically relevant conditions. Methods: We studied the effects of tiplaxtinin on migration of smooth muscle cells (SMCs) and endothelial cells (ECs). Results: Tiplaxtinin significantly inhibited migration of murine SMCs through 3-dimensional (3-D) collagen matrix in a concentration-dependent manner. Tiplaxtinin did not inhibit SMC proliferation, and it did not inhibit migration of PAI-1-deficient SMCs, suggesting that tiplaxtinin’s effect on SMCs was non-toxic and PAI-1-dependent. The anti-migratory effect of tiplaxtinin on SMCs was preserved in collagen 3-D matrix containing vitronectin and other extracellular matrix molecules, further supporting the physiological significance of the effect. In contrast to SMCs, tiplaxtinin did not inhibit migration of human aortic ECs in vitro or murine ECs in vivo, the latter assessed in a murine carotid injury model. To study the basis for the differential effect of tiplaxtinin on SMCs vs. ECs, we compared expression of LDL receptor-related protein 1 (LRP1), a motogenic receptor for PAI-1, between cell types by RT-PCR and found that LRP1 gene expression was significantly lower in ECs than in SMCs. Furthermore, recombinant PAI-1 stimulated the migration of wild-type mouse embryonic fibroblasts (MEFs), but not LRP1-deficient MEFs. Conclusions: Tiplaxtinin, a pharmacological inhibitor of PAI-1, inhibits SMC migration under physiological conditions, while having no inhibitory effect on EC migration. The differential effect of PAI-1 inhibition on SMCs vs. ECs appears to be mediated by LRP1 and may be of clinical significance, as it is advantageous to prevent intimal hyperplasia by inhibiting SMC migration without inhibiting EC migration, which is key to preserving an intact, anti-thrombotic vascular endothelium.