Erin M. Lynch

Targeted Deletion of CCR2 Impairs Deep Vein Thombosis Resolution in a Mouse Model

CCR2 is required for monocyte recruitment in many inflammatory processes, as well as conferring Th1 lymphokine responses. Deep vein thrombosis (DVT) resolution represents a specific inflammatory response whereby the thrombus must be dissolved for restoration of blood flow. Using a stasis model of DVT in the mouse, we investigated the role of CCR2 on DVT resolution. Genetic deletion of CCR2 (CCR2−/−) was associated with larger thrombi at early and later time points, increased thrombus collagen, fewer thrombus monocytes (F4/80), and significantly impaired neovascularization. IL-2 and IFN-γ were significantly reduced in early CCR2−/− thrombi, whereas MCP-1 was significantly increased, and Th2 lymphokines were unaffected. Supplementation of CCR2−/− mice with IFN-γ normalized early thrombus resolution without increasing monocyte influx. Neither Ab depletion of IFN-γ nor genetic deletion of IFN-γ impaired early DVT resolution. Early fibrinolysis was not impaired in CCR2−/− mice, but a significant reduction in both matrix metalloproteinase (MMP)-2 and MMP-9 activity was observed. However, only MMP-9 activity was restored with administration of IFN-γ. We conclude that an early CCR2-dependent Th1 lymphokine response predominates in normal DVT resolution, mediates this in part by MMP-9 activation, but is not solely dependent on IFN-γ.

Fibrotic injury after experimental deep vein thrombosis is determined by the mechanism of thrombogenesis

Vessel wall matrix changes occur after injury, although this has not been well studied in the venous system. This study tested the hypothesis that the thrombus dictates the vein wall response and vein wall damage is directly related to the duration of thrombus contact. To determine the injury response over time, rats underwent inferior vena cava (IVC) ligation to produce a stasis thrombus, with harvest at various time points to 28 days (d). Significant vein wall matrix changes occurred with biomechanical injury (stiffness) peaking at 7-14 d, with concurrent early reduction in total collagen, an increase in early matrix metalloproteinase (MMP)-9 and late MMP-2, and concomitant increase in tumor necrosis factor (TNF)alpha, monocyte chemoattractant(MCP)-1 and tumor growth factor (TGF)beta (all P<0.05). To isolate the effect of the thrombus and its mechanism of genesis, rats underwent 7 d or limited stasis (24 hours), non-stasis thrombosis, or non-thrombotic IVC occlusion (Silicone plug). Vein wall stiffness was increased seven-fold, with a five-fold reduction in collagen, and 5.5- to seven-fold increase in TNFalpha, MCP-1, and TGFbeta with 7 d stasis as compared with controls (all P<0.05). By Picosirus red staining analysis, collagenolysis was significantly greater with 7 d stasis injury (P=0.01) but neither MMP-9 nor MMP-2 activity correlated with injury mechanism. In addition, vein wall cellular proliferation and uPA gene expression paralled the stasis thrombotic injury. Limited stasis, non-stasis thrombosis and non-thrombotic IVC occlusion showed a lesser inflammatory response. These data suggest both a static component and the thrombus directs vein wall injury via multiple mechanisms.

Neutrophils modulate post-thrombotic vein wall remodeling but not thrombus neovascularization

Early deep venous thrombosis (DVT) resolution is associated with neutrophil (PMN) influx. This study examined the role of PMNs in thrombus neovascularization and vein wall injury after DVT. A rat model of DVT by inferior vena cava (IVC) ligation was performed with control serum or rabbit anti-rat PMN serum administered perioperatively with sacrifice at 2 and 7 days. At 2 days, neutropenic rats had 1.6-fold larger thrombi (P = .04) and 1.4-fold higher femoral venous pressures by water manometry (P = .008) but no difference in thrombus neovascularization was observed. By 7 days, DVT sizes were similar, but vein wall injury persisted in the neutropenic rats with a 2.0-fold increase in vein wall stiffness by microtensiometry (P < .05), as well as a 1.2-fold increased thickness (P = .04). Collagen and profibrotic growth factors were significantly increased in neutropenic IVC at 7 days (all P < .05). Vein wall and intrathrombus uPA byWestern immunoblotting, and intrathrombus MMP-9 gelatinase activity were significantly less in neutropenic rats than controls (P < .001). Conversely, MMP-2 was significantly elevated in neutropenic IVC at 2 days after DVT. However, neutropenia induced 24 hours after DVT formation resulted in no significant increase in vein wall stiffness or collagen levels at 7 days, despite 1.4-fold larger thrombi (P < .05). These data suggest a critical early role for PMN in post DVT vein wall remodeling.

Vein wall re-endothelialization after deep vein thrombosis is improved with low-molecular-weight heparin.

OBJECTIVE Vein wall endothelial turnover after stasis deep vein thrombosis (DVT) has not been well characterized. The purpose of this study was to quantify re-endothelialization after DVT and determine if low-molecular-weight heparin (LMWH) therapy affects this process. METHODS Stasis DVT was generated in the rat by inferior vena cava ligation, with harvest at 1, 4, and 14 days. Immunohistologic quantification of vascular smooth muscle cells and luminal endothelialization was estimated by positive staining for alpha-smooth muscle actin and von Willebrand factor, respectively. In separate experiments, rats were treated either before or after DVT with subcutaneous LMWH (3 mg/kg daily) until harvesting at 4 and 14 days. The inferior vena cava was processed for histologic analysis or was processed for organ culture after the thrombus was gently removed. The vein wall was stimulated in vitro with interleukin-1beta (1 ng/mL), and the supernatant was processed at 48 hours for nitric oxide. Cells were processed by real-time polymerase chain reaction for endothelial nitric oxide synthase, inducible nitric oxide synthase, cyclooxygenase-1 and -2, and thrombomodulin at 4 and 14 days, and collagen I and III at 14 days. Comparisons were done with analysis of variance or t test. A P < .05 was significant. RESULTS Thrombus size peaked at 4 days, whereas luminal re-endothelialization increased over time (1 day, 11% +/- 2%; 4 days, 23% +/- 4%; 14 days, 64% +/- 7% (+) von Willebrand factor staining; P < .01, n = 3 to 4, compared with non-DVT control). Similarly, vascular smooth muscle cell staining was lowest at day 1 and gradually returned to baseline by 14 days. Both before and after DVT, LMWH significantly increased luminal re-endothelialization, without a difference in thrombus size at 4 days, but no significant difference was noted at 14 days despite smaller thrombi with LMWH treatment. Pretreatment with LMWH was associated with increased vascular smooth muscle cell area and recovery of certain inducible endothelial specific genes. No significant difference in nitric oxide levels in the supernatant was found at 4 days. At 14 days, type III collagen was significantly elevated with LMWH treatment. CONCLUSIONS Venous re-endothelialization occurs progressively as the DVT resolves and can be accelerated with LMWH treatment, although this effect appears limited to the early time frame. These findings may have clinical relevance for LMWH timing and treatment compared with mechanical forms of therapy. CLINICAL RELEVANCE How the vein wall endothelium responds after deep vein thrombosis (DVT) has not been well documented owing to limited human specimens. This report shows that low-molecular-weight heparin accelerates or protects the endothelium and preserves medial smooth muscle cell integrity after DVT, but that this effect is limited to a relatively early time period. Although most DVT prophylaxis is pharmacologic (a heparin agent), use of nonpharmacologic measures is also common. The use of heparin prophylaxis, compared with after DVT treatment, and the acceleration of post-DVT re-endothelialization require clinical correlation.

Meteoric fluid infiltration in crustal-scale normal fault systems as indicated by δ18O and δ2H geochemistry and 40Ar/39Ar dating of neoformed clays in brittle fault rocks

Both the sources and pathways of fluid circulation are key factors to understanding the evolution of low-angle normal fault (LANF) systems and the distribution of mineral deposits in the upper crust. In recent years, several reports have shown the presence of meteoric waters in mylonitic LANF systems at mid-crustal conditions. However, a mechanism for meteoric water infiltration to these mid-crustal depths is not well understood. Here we report paired δ 18 O and δ 2 H isotopic values from dated, neoformed clays in fault gouge in major detachments of the southwest United States. These isotopic values demonstrate that brittle fault rocks formed from exchange with pristine to weakly evolved meteoric waters at multiple depths along the detachment. 40 Ar/ 39 Ar dating of these same neoformed clays constrains the Pliocene ages of fault-gouge formation in the Death Valley area. The infiltration of ancient meteoric fluids to multiple depths in LANFs indicates that crustal-scale normal fault systems are highly permeable on geologic timescales and that they are conduits for efficient, coupled flow of surface fluids to depths of the brittle-plastic transition.