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.

Apoptosis of hepatocytes is a seminal feature of fulminant hepatic failure. We show that the anti-apoptotic protein A20 is upregulated in hepatocytes by pro-inflammatory stimuli and functions to protect from apoptosis and limit inflammation by inhibiting NF-B. Adenoviral mediated hepatic expression of A20 in BALB/c mice yields an 85% survival rate in the D-galactosamine (D-gal)/lipolysaccharide (LPS) model of acute toxic hepatitis compared with 15% to 20 % in control mice. Expression of A20 preserves normal liver function as assessed by prothrombin time. The protective effect of A20 is independent of tumor necrosis factor (TNF) inhibition. Maintaining high circulating TNF levels may be advantageous for liver regeneration. Our data supports this hypothesis as evidenced by increased proliferating cell nuclear antigen (PCNA) expression in the livers of mice expressing A20 compared with a dominant negative mutant of the TNF receptor (TNF-R), 6 hours following D-gal/LPS administration. In conclusion, these results qualify A20 as part of a physiologic, protective response of hepatocytes to injury and a promising gene therapy candidate for clinical applications aimed at preventing and treating viral and toxic fulminant hepatic failure. I n normal livers, apoptosis is a rare event (2-4 apoptotic cells/ 10,000) reflecting physiologic cell turnover. 1 Necrosis and apoptosis of hepatocytes are critical pathologic features of liver injury. 1 Hepatotoxic substances such as ethanol, acetaminophen, and cytostatic drugs result in hepatocyte apoptosis. 1 Apoptosis of hepatocytes is also a feature of viral hepatitis, ischemic liver injury, sepsis, and cholestasis. 2,3 Hepatocyte apoptosis and necrosis, when massive, result in fulminant hepatic failure (FHF). 4 Only 14% of patients diagnosed with FHF recover with medical therapy. Orthotopic liver transplantation (OLT) has improved the fate of these patients (49% undergo OLT), yet 37% die while awaiting OLT. 5 OLT carries high surgical, infectious and oncogenic risks associated with immunosuppression. 5 Identifying and increasing the anti-apoptotic armamentarium of hepatocytes is one approach to treating FHF. Hepatocytes express lower constitutive levels of Bcl-2 and Bcl-x L (the prototypic bcl family members) than other organs, which could account for their particular sensitivity to Fas-mediated apoptosis. 6 Expression of a bcl-2 or a bcl-x L transgene in hepatocytes protects mice from anti-Fas-induced FHF but not against acute toxic hepatitis induced by D-galactosamine (D-gal) and lipopolysaccharide (LPS) or TNF, prompting us to seek other protective genes. 7,8 In this study, we sought to determine whether the anti-apoptotic protein A20 is expressed in hepatocytes and fills a cytoprotective function. We were particularly interested in whether A20 could protect mice from D-gal/LPS acute toxic hepatitis, a model in which Bcl family members are ineffective. 9 Combined administration of LPS and D-gal show biochemical and metabolic changes akin to FHF. 10 A20 was originally identified...

A20 is a stress response gene in endothelial cells (ECs). A20 serves a dual cytoprotective function, protecting from tumor necrosis factor (TNF)-mediated apoptosis and inhibiting inflammation via blockade of the transcription factor nuclear factor-B (NF-B). In this study, we evaluated the molecular basis of the cytoprotective function of A20 in EC cultures and questioned whether its protective effect extends beyond TNF to other apoptotic and necrotic stimuli. Our data demonstrate that A20 targets the TNF apoptotic pathway by inhibiting proteolytic cleavage of apical caspases 8 and 2, executioner caspases 3 and 6, Bid cleavage, and release of cytochrome c, thus preserving mitochondrion integrity. A20 also protects from Fas/CD95 and significantly blunts natural killer cell-mediated EC apoptosis by inhibiting caspase 8 activation. In addition to protecting ECs from apoptotic stimuli, A20 safeguards ECs from complement-mediated necrosis. These data demonstrate, for the first time, that the cytoprotective effect of A20 in ECs is not limited to TNF-triggered apoptosis. Rather, A20 affords broad EC protective functions by effectively shutting down cell death pathways initiated by inflammatory and immune offenders. IntroductionA20 is a zinc finger protein originally identified as a tumor necrosis factor (TNF)-responsive gene in endothelial cells (ECs). 1 A20 is expressed in multiple cell types in response to a variety of stimuli that activate the transcription factor nuclear factor-B (NF-B), including interleukin 1 (IL-1), lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), H 2 O 2 , and CD40 ligand. [2][3][4][5][6][7] We and others have demonstrated that A20, initially described as an antiapoptotic gene, is also a potent inhibitor of the transcription factor NF-B. 7-9 A20-null mice fail to terminate TNF-induced NF-B activation, develop severe inflammation and cachexia, and die prematurely, indicating the importance of A20 in the hierarchy of anti-inflammatory defense processes. 6,10 Elucidating the molecular basis and binding partner(s) that determine the inhibitory effect of A20 upon NF-B activation is the focus of ongoing research. 8 A20 has been shown to interact with components of the NF-B signaling cascade upstream of inhibitor of NF-B (IB), including TNF receptor-associated factors (TRAFs) 11,12 TRAF-1, TRAF-2, and TRAF-6; A20 binding inhibitors of NF-B; and the signalosome IB kinase-␥ (IKK␥)/NF-B essential modulator (NEMO) unit. [13][14][15] Further work is required to test the relevance of these interactions in blocking NF-B activation in response to stimuli other than TNF. 16 In contrast to its so far universal inhibitory effect on NF-B activation, the antiapoptotic activity of A20 remains controversial and appears to be specific to cell type and stimulus. Overexpression of A20 protects human breast carcinoma MCF-7 cells, murine fibrosarcoma WEHI 164, and murine embryonic NIH3T3, but not Hela and lung epithelial A459 cells from TNF-mediated apoptosis. 9,[17][18][19] In cultures derived from prima...

Transplantation of an excessive number of islets of Langerhans (two to four pancreata per recipient) into patients with type I diabetes is required to restore euglycemia. Hypoxia, nutrient deprivation, local inflammation, and the β cell inflammatory response (up-regulation of NF-κB-dependent genes such as inos) result in β cell destruction in the early post-transplantation period. Genetic engineering of islets with anti-inflammatory and antiapoptotic genes may prevent β cell loss and primary nonfunction. We have shown in vitro that A20 inhibits NF-κB activation in islets and protects from cytokine- and death receptor-mediated apoptosis. In vivo, protection of newly transplanted islets would reduce the number of islets required for successful transplantation. Transplantation of 500 B6/AF1 mouse islets into syngeneic, diabetic recipients resulted in a cure rate of 100% within 5 days. Transplantation of 250 islets resulted in a cure rate of only 20%. Transplantation of 250 islets overexpressing A20 resulted in a cure rate of 75% with a mean time to cure of 5.2 days, comparable to that achieved with 500 islets. A20-expressing islets preserve functional β cell mass and are protected from cell death. These data demonstrate that A20 is an ideal cytoprotective gene therapy candidate for islet transplantation.

A20 is a NF-kappaB-dependent gene that has dual anti-inflammatory and antiapoptotic functions in endothelial cells (EC). The function of A20 in smooth muscle cells (SMC) is unknown. We demonstrate that A20 is induced in SMC in response to inflammatory stimuli and serves an anti-inflammatory function via blockade of NF-kappaB and NF-kappaB-dependent proteins ICAM-1 and MCP-1. A20 inhibits SMC proliferation via increased expression of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Surprisingly, A20 sensitizes SMC to cytokine- and Fas-mediated apoptosis through a novel NO-dependent mechanism. In vivo, adenoviral delivery of A20 to medial rat carotid artery SMC after balloon angioplasty prevents neointimal hyperplasia by blocking SMC proliferation and accelerating re-endothelialization, without causing apoptosis. However, expression of A20 in established neointimal lesions leads to their regression through increased apoptosis. This is the first demonstration that A20 exerts two levels of control of vascular remodeling and healing. A20 prevents neointimal hyperplasia through combined anti-inflammatory and antiproliferative functions in medial SMC. If SMC evade this first barrier and neointima is formed, A20 has a therapeutic potential by uniquely sensitizing neointimal SMC to apoptosis. A20-based therapies hold promise for the prevention and treatment of neointimal disease.

The liver has a remarkable regenerative capacity, allowing recovery following injury. Regeneration after injury is contingent on maintenance of healthy residual liver mass, otherwise fulminant hepatic failure (FHF) may arise. Understanding the protective mechanisms safeguarding hepatocytes and promoting their proliferation is critical for devising therapeutic strategies for FHF. We demonstrate that A20 is part of the physiological response of hepatocytes to injury. In particular, A20 is significantly upregulated in the liver following partial hepatectomy. A20 protects hepatocytes from apoptosis and ongoing inflammation by inhibiting NF-B. Hepatic expression of A20 in BALB/c mice dramatically improves survival following extended and radical lethal hepatectomy. A20 expression in the liver limits hepatocellular damage hence maintains bilirubin clearance and the liver synthetic function. In addition, A20 confers a proliferative advantage to hepatocytes via decreased expression of the cyclin-dependent kinase inhibitor p21 waf1 . In conclusion, A20 provides a proliferative advantage to hepatocytes. By combining anti-inflammatory, antiapoptotic and pro-proliferative functions, A20-based therapies could be beneficial in prevention and treatment of FHF. (HEPATOLOGY 2005;42:156-164.)

Current plasma markers for diagnosis of deep venous thrombosis (DVT) allow for exclusion of the diagnosis, but lack adequate specificity to establish the diagnosis. Thus, a prospective study was performed to determine the sensitivity and specificity of plasma assays for D-dimer, soluble P-selectin (P-selectin), and total microparticles in patients with documented DVT by duplex ultrasound. Three groups of individuals were examined: 30 normals; 22 positive for DVT on duplex ultrasound (Group 2); and 21 symptomatic, but negative on duplex ultrasound for DVT (Group 3). Group 1 individuals had D-dimer values of 1.53 +/- 0.12 mg/l and P-selectin values of 0.34 +/- 0.05 ng/mg total protein. Group 2 vs. Group 3 individuals had D-dimer values of 7.57 +/- 2.03 vs. 3.19 +/- 0.79 mg/l, p = 0.02; P-selectin values of 0.98 +/- 0.11 vs. 0.55 +/- 0.08 ng/mg total protein, p < 0.01; and micro-particle values of 129 +/- 17% vs. 99 +/- 12% of control, p = ns. Using a logistic regression model with dichotomous variables, we determined a sensitivity of 73%, specificity of 81%, and accuracy of 77% when combining D-dimer, soluble P-selectin, and total microparticles to differentiate Group 2 from Group 3 patients. Logistic regression using continuous variables yielded similar results (p = 0.05). This study demonstrates that plasma markers for DVT can be developed and achieve moderate sensitivity and specificity in diagnosing DVT. However for clinical applicability, the sensitivity/specificity will need to be improved. These studies also suggest the importance of soluble P-selectin in assessing DVT in humans.

Background-CD40/CD40 ligand (CD40L) signaling is a potent activator of endothelial cells (ECs) and promoter of atherosclerosis. In this study, we investigate whether A20 (a gene we have shown to be antiinflammatory and antiapoptotic in ECs) can protect from CD40/CD40L-mediated EC activation. Methods and Results-Overexpression of CD40, in a transient transfection system, activates the transcription factor NF-B and upregulates IB␣, E-selectin, and tissue factor (TF) reporter activity. Coexpression of A20 inhibits NF-B and upregulation of IB␣ and E-Selectin but not TF, suggesting that CD40 induces TF in a non-NF-B-dependent manner. In human coronary artery ECs (HCAECs), adenovirus-mediated overexpression of A20 blocks physiological, CD40-induced activation of NF-B, upstream of IB␣ degradation (Western blot) and subsequently upregulation of ICAM-1, VCAM-1, and E-selectin (flow cytometry). Although A20 does not block TF transcription its expression in HCAECs inhibits TF induction (colorimetric assay and RT-PCR) by blunting CD40 upregulation. We demonstrate that CD40 signaling induces apoptosis in a proinflammatory microenvironment. A20 overexpression protects from CD40-mediated EC apoptosis (DNA content analysis and trypan blue exclusion). We also demonstrate that signaling through CD40L activates NF-B and induces apoptosis in ECs, both of which are inhibited by A20 overexpression. Conclusion-A20 works at multiple levels to protect ECs from CD40/CD40L mediated activation and apoptosis.A20-based therapy could be beneficial for the treatment of vascular diseases such as atherosclerosis and transplantassociated vasculopathy.