Effect of TNFα blockade on UVB-induced inflammatory cell migration and collagen loss in mice.

UVB irradiation induces pro-inflammatory cytokines including interleukin-1 (IL-1) and tumor necrosis factor-α (TNFα) in the skin. TNFα stimulates the chemotaxis of inflammatory cells to the skin. These cells secrete metalloproteinases (MMPs) and other enzymes that damage the cutaneous matrix. Therefore, blocking TNFα activity could be effective in preventing the influx of inflammatory cells and subsequent collagen degradation in the skin. In addition, TNFα downregulates procollagen mRNA, and thus blockade may be beneficial to production of type I collagen. Female C57BL/6 J mice were treated with etanercept (TNFα blocker, 4 mg/kg/day) for 4 days 1 h prior to UVB irradiation (100 mJ/cm2/day for 5 days). On the 5th day mice were sacrificed 3 h after UVB exposure. Blocking TNFα significantly inhibited UVB-induced recruitment of macrophages, mast cells, and neutrophils. UVB-irradiated mice skin contained more mature collagen compared to etanercept and UVB + etanercept-treated mice. Skin from UVB + etanercept-treated mice had more collagen fragments relative to UVB-irradiated mice. Procollagen protein was lower in UVB-irradiated and UVB + etanercept-treated mice. TNFα blockade decreased decorin and TGF-ß1 in UVB-irradiated mice compared to UVB alone. MMP13 was inhibited by etanercept in UVB-irradiated mice (p < 0.01). In conclusion, blockade of TNFα significantly decreased mature collagen in UVB-irradiated mice, while increasing collagen fragmentation and decreasing procollagen.

Intrasplenic transplantation of isolated adult rat hepatocytes: sex-reversal and/or suppression of the major constituent isoforms of cytochrome P450.

Adult male and female rat hepatocytes were individually transplanted into the spleens of adult male and female rats. The recipients were euthanized at either eight, sixteen, thirty, or forty-five weeks following transplantation, at which time hepatic and splenic levels of liver-specific rat albumin mRNA as well as sex-dependent transcript levels of CYP2C11, -2C12, -2C7, -2A1, and -3A2-which accounts for > 60% of the total concentration of hepatic constituent cytochrome P450-were determined. Whereas the pre-infused hepatocytes expressed their expected cytochrome P450 sexual dimorphisms (female-specific CYP2C12, male-specific CYP3A2, and female-predominant CYP2A1), their post-transplantational competence now reflected the sexual dimorphisms of the recipient (as observed in the host's liver), which supports the concept that the sex-dependent growth hormone circulating profiles are the determinants regulating the expression levels of hepatic cytochrome P450. Also expressed at normal concentrations in the pre-infused hepatocytes, male-specific CYP2C11 and female-predominant CYP2C7 were inexplicably undetectable in the spleens of both recipient males and females, regardless of the sex of the donor hepatocytes, almost one year after transplantation.

Animal models of acute photodamage: comparisons of anatomic, cellular and molecular responses in C57BL/6J, SKH1 and Balb/c mice.

Human cutaneous photodamage is a major medical problem that includes premature aging and fragility of the skin. Nonxenografted animal models have not been comparatively evaluated for how well they resemble the changes seen in human skin. Here, we sought to identify a suitable mouse model that recapitulates key anatomic, cellular and molecular responses observed in human skin during acute UV exposure. Adult females from three strains of mice, C57BL/6J, SKH1 and Balb/c were exposed to UVB and then evaluated 3 or 20 h after the last irradiation. Skin from UVB-exposed C57BL/6J mice showed features resembling human photodamage, including epidermal thickening, infiltration of the dermis with inflammatory cells, induction of tumor necrosis factor-α (TNF-α) mRNA, accumulation of glycosaminoglycans, particularly hyaluronan in the epidermis and loss of collagen. Hairless SKH1 mouse skin responded similarly, but without any induction of TNF-α mRNA or chondroitin sulfate. Irradiated Balb/c mice were the least similar to humans. Our results in C57BL/6J mice and to a lesser extent in SKH1 mice, show cutaneous responses to a course of UVB-irradiation that mirror those seen in human skin. Proper choice of model is critical for investigating cellular and molecular mechanisms of photodamage and photoaging.

UVB and proinflammatory cytokines synergistically activate TNF-alpha production in keratinocytes through enhanced gene transcription.

UVB irradiation potently induces cytokines in the skin, including IL-1alpha and tumor necrosis factor-alpha (TNF-alpha). The mechanism for TNF-alpha induction in UVB-irradiated keratinocytes is not clear. In this study, we explored the effects of UVB and cytokines, alone or in combination in human keratinocytes. Keratinocytes were sham- or UVB-irradiated with 30 mJ cm(-2), and then incubated in the absence or presence of IFN-alpha2b, TNF-alpha, or IL-1alpha. UVB and IL-1alpha treatment synergistically enhanced TNF-alpha secretion and mRNA levels in human keratinocytes, similar to the findings reported previously in human fibroblasts. Exogenous recombinant TNF-alpha up-regulates its own mRNA level. However, addition of IFN-alpha2b did not show any additive effect on TNF-alpha mRNA induction. To understand the regulation of TNF-alpha mRNA by UVB, with or without IL-1alpha, we examined the transcription rate and half-life of TNF-alpha mRNA. Treatment of keratinocytes with IL-1alpha or UVB alone increased TNF-alpha gene transcription 4- to 5-fold over sham treatment, and TNF-alpha gene transcription increased 11-fold in cells treated with UVB plus IL-1alpha over sham. UVB with IL-1alpha did not enhance the half-life of TNF-alpha mRNA over that seen with UVB alone. In conclusion, TNF-alpha expression in primary keratinocytes is upregulated transcriptionally by UVB and IL-1alpha.

Increased severity of alcoholic liver injury in female verses male rats: a microarray analysis.

Alcoholic liver disease (ALD) is an increasingly recognized condition that may progress to end-stage liver disease. In addition to alcohol consumption, genetic factors, dietary fatty acids, gender and viral infection potentiate the severity of alcoholic liver injury. In humans, significant gender differences in susceptibility to ALD are observed. In the intragastric infusion rat model of ALD, female rats developed more severe liver injury than males. To understand the effect of gender on the development of more severe ALD in female rats, we performed a microarray based expression profiling of genes in rats fed with fish oil and ethanol diet. A large number of genes showed significant changes in female livers compared to males. The upregulated genes in female liver were involved in proteosome endopeptidase activity, catalytic activity, lipid metabolism, alcohol metabolism, mitochondrial and oxidoreductase activity. The downregulated genes were involved in oxidoreductase activity, chaperone activity, and electron transport activity in the female liver as demonstrated by biological theme analysis. Ingenuity computational pathway analysis tools were used to identify specific regulatory networks of genes operative in promoting liver injury. These networks allowed us to identify a large cluster of genes involved in lipid metabolism, development, cellular growth and proliferation, apoptosis, carcinogenesis and various signaling pathways. Genes listed in this article that were significantly increased or decreased (expression two fold or more) were assigned to pathological functional groups and reviewed for relevance to establish hypotheses of potential mechanisms involved in ALD in female liver injury.

Transcriptional networks in a rat model for nonalcoholic fatty liver disease: a microarray analysis.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a common hepatic condition that may progress to end-stage liver disease. High-fat diets in animals reproduce many of the features found in nonalcoholic steatohepatitis. OBJECTIVE: To understand how various dietary or genetic factors influence the development of fatty liver and consequently NAFLD, we performed microarray-based expression profiling of genes, induced by fish oil and dextrose diet, a putative mediator of alcohol-like effects on the liver of the female rat. DESIGN: Male and age-matched female rats were fed fish oil and dextrose for 4 weeks. Hepatic RNA from each sample was extracted and used for microarray analysis. RESULTS: A large number of genes underwent significant changes in the female liver as compared to male controls. In the female rat liver, biological theme analysis demonstrated a shift in the transcriptional program which included upregulation of genes involved in lipid metabolism, chaperone activity, mitochondrial and oxidoreductase activity combined with downregulation of genes involved in nucleic acid metabolism. The differential expression of genes of interest identified by microarray technique was validated by real-time reverse transcription-polymerase chain reaction. Ingenuity computational pathway analysis tools were used to identify specific regulatory networks of genes operative in promoting liver injury. CONCLUSIONS: The use of networks stated above allowed us to identify genes involved in cell death, apoptosis, peroxisome proliferator-activated receptor alpha-regulated lipid metabolism and mitogen-activated protein kinase signaling pathways.

Antibody-directed targeting of angiostatin's receptor annexin II inhibits Lewis Lung Carcinoma tumor growth via blocking of plasminogen activation: possible biochemical mechanism of angiostatin's action.

Angiostatin, the N-terminal four kringles (K1-4) of parent molecule plasminogen, is reported to block Lewis Lung Carcinoma (LLC) tumor growth and metastasis. However, angiostatin's mechanism of action is unclear. We earlier reported that angiostatin binds to cell surface annexin II through the lysine-binding domain (kringles 1-4) [Tuszynski, G.P., Sharma, M., Rothman, V.L., Sharma, M.C., 2002. Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells. Microvasc. Res. 64:448-462.]). We now show that annexin II on the cell surface of LLC cells regulates conversion of plasminogen to plasmin. Activation of plasminogen to plasmin is time-dependent, with the linear activation lasting up to 120 min. Monoclonal antibodies to annexin II reduced plasminogen activation by 92.6%, suggesting a specific role of annexin II in plasmin generation. Angiostatin also reduced plasmin generation by 81.6%, suggesting that angiostatin may be competing with plasminogen through lysine-binding domain. epsilon-Aminocaproic acid, a lysine analogue, effectively blocked plasminogen activation indicating that, indeed, the lysine-binding site of the kringles domain is required for activation. These data suggest that annexin II may be a receptor target for angiostatin's action. Therefore, we tested the effect of high affinity monoclonal antibody to annexin II in mouse model of LLC. A single dose of antibody treatment inhibited LLC tumor growth almost 70% with concomitant inhibition of circulating plasmin generation and its proteolytic activity. Taken together, it is possible that inhibition of LLC tumor growth and metastasis reported by angiostatin therapy may be due to blocking of annexin-II-dependent plasmin generation. Plasmin is known to influence angiogenic, invasive and metastatic capability of tumors.

Angiogenesis-associated protein annexin II in breast cancer: selective expression in invasive breast cancer and contribution to tumor invasion and progression.

Many advanced human tumors including breast cancer overproduce plasmin that is known to promote angiogenesis and metastasis. The mechanism of this effect is poorly understood. Here we report that annexin II, an endothelial co-receptor for tPA (tissue-type plasminogen activator) and plasminogen, was undetectable in normal and hyperplastic ductal epithelial cells and ductal complexes. By contrast, it was consistently expressed in invasive breast cancer and ductal carcinoma in situ (DCIS) indicating its involvement in breast cancer. Using the well established invasive/metastatic MDA-MB231 cell line and the noninvasive/nonmetastatic MCF-7 human breast cancer cell line, we investigated the mechanism by which annexin II regulates breast cancer progression and metastasis. Western and Northern blot analyses demonstrate selective expression of annexin II in MDA-MB231 cells but not in poorly invasive MCF-7 cells suggesting its participation in invasive breast cancer. Since annexin II is a receptor for plasminogen, we tested whether MDA-MB231 cells are capable of producing plasmin in vitro. MDA-MB231 cell membranes induced plasmin generation in a time-dependent manner while those from MCF-7 cells failed to convert plasminogen to plasmin. The generated plasmin is capable of degrading ECM consequently facilitating cell invasion and migration, biological functions required for angiogenesis and metastasis. Plasmin generation and its dependent invasion and migration can be blocked by a monoclonal antibody to annexin II or angiostatin, potent inhibitors of angiogenesis, breast cancer, and metastasis. Our findings indicate that annexin II-dependent localized plasmin generation by human breast cancer cells could contribute to angiogenesis and metastasis. These results suggest that annexin II may be an attractive target for new anti-angiogenic and anti-breast cancer therapies.

Angiostatin-induced inhibition of endothelial cell proliferation/apoptosis is associated with the down-regulation of cell cycle regulatory protein cdk5.

Endothelial cells (ECs) are quiescent in normal blood vessels, but undergo rapid bursts of proliferation after vascular injury, hypoxia or induced by powerful angiogenic cytokines like fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Deregulated proliferation of ECs facilitates angiogenic processes and promotes tumor growth. In dividing cells, cell cycle-associated protein kinases, which are referred as cyclin-dependent kinases (cdks), regulate proliferation, differentiation, senescence, and apoptosis. Cyclin-dependent kinase-5 (cdk5) is expressed in neuronal cells and plays an important role in neurite outgrowth, of neuronal migration and neurogenesis, its functions in non-neuronal cells are unclear. Here, we show for the first time that the cdk5 is expressed at high levels in proliferating bovine aortic endothelial (BAE) cells, by contrast insignificant low levels of cdk5 expression in quiescent BAE cells. In addition, bFGF up-regulates cdk5 expression in a dose-dependent fashion. Interestingly, temporal expression data suggests that cdk5 expression is very low between 24-48 h, but high level of cdk5 expression was detected during 60-72 h. This later time corresponds to the time of completion of one cell cycle (doubling of cell population) of BAE cell culture. Angiostatin (AS), a powerful inhibitor of angiogenesis inhibits ECs proliferation in dose-dependent manner with concomitant down-regulation of cdk5 expression. The role of cdk5 in ECs, proliferation and apoptosis was confirmed by selective inhibition of cdk5 expression by the purine derivative roscovitine, which inhibits bFGF-stimulated BAE cells proliferation and induces apoptosis in dose-specific manner. By contrast, the roscovitine analog olomoucine, which is a specific inhibitor of cdk4, but not of cdk5 failed to affect ECs proliferation and apoptosis. These data suggest for the first time that neuron specific protein cdk5 may have significant role in the regulation of ECs proliferation, apoptosis, and angiogenesis and extends beyond its role in neurogenesis.

Spurious observation of splenic cyp2b1 expression.

Phenobarbital (PB) induction of the CYP2B subfamily was studied in the livers and spleens of male and female rats. Animals were treated with either PB (10 mg/kg) or vehicle for 4 consecutive days. A reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative Northern blotting, Western blotting, and a radioenzymatic assay were used to observe differential levels of CYP2B1 and CYP2B2 mRNAs, proteins, and catalytic activities. CYP2B2 expression was limited to the livers of PB-treated male and female rats and was not detected in spleen. Low constitutive levels of CYP2B1 mRNA were markedly induced approximately 7- to 17-fold in the livers of PB-treated male and female rats, respectively. However, using the same standard oligonucleotide probe for CYP2B1 mRNA, we observed considerably greater constitutive concentrations of the transcript in spleen than in liver. Putative splenic CYP2B1 mRNA was significantly elevated by the PB treatment, although not as profoundly as the hepatic response. In contrast, only the livers of the barbiturate-treated rats expressed CYP2B1 proteins or specific catalytic activity (androstenedione 16beta-hydroxylase). Protein and catalytic activities of the isoforms were undetectable in spleen of either male or female vehicle- and PB-treated rats. In agreement, RT-PCR was unable to demonstrate the expression of splenic CYP2B1 mRNAs. Investigating the possibility that the Northern probe for CYP2B1 was identifying a similar sequence isoform, we performed RT-PCR using primers for CYP2B12 and CYP2B15. Since neither of these isoforms was expressed in spleen, we conclude that the spurious results using the Northern probe for CYP2B1 mRNA were due to the presence of a cross-reacting, PB-responsive transcript not currently identifiable in existing databases.

Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells.

Angiostatin(AS), an internal fragment of plasminogen, is one of the most potent specific inhibitors of angiogenesis. Angiostatin treatment has resulted in the complete regression of human tumors implanted subcutaneously into nude mice and has great therapeutic value (O'Reilly et al., Nat. Med. 2, 689-692, 1996). Despite promising therapeutic value in the treatment of cancer, the mechanism of its action is still unknown. We found that angiostatin binds to a 35-kDa protein in bovine aortic endothelial (BAE) cells (Sharma et al., Proc. Am. Assoc. Cancer Res. 42, 568, A3050, 2002). In an attempt to begin to understand angiostatin's mechanism of action, we have purified and characterized this 35-kDa protein from BAE cells. Internal peptide sequence analysis of purified protein demonstrated (SLYYIQQDTK, SYSPYDMLESIK, and ALLYLXGGDD) 100% sequence identity with tyrosine kinase substrate annexin II. Solid phase binding analysis suggests that angiostatin specifically bound to purified annexin II immobilized on 96-well plastic plates. Hundred-fold molar excess of unlabeled AS and anti-annexin II antibody inhibited bindings 85 and 55%, respectively, suggesting specific interaction. Annexin II is a predominant receptor for angiostatin, since neutralizing the angiostatin by soluble receptor (annexin II) effectively blocks angiostatin's anti-EC activity. Similarly, saturating the annexin II receptor by plasminogen in endothelial cells also blocks angiostatin's activity. Both angiostatin and plasminogen bind to purified annexin II in BAE cells saturably with apparent K(d) values of 101 and 164 nM, respectively, for purified annexin II and K(d) values of 83 and 125 nM, respectively, for BAE cells. Anti-annexin II monoclonal antibody inhibited angiostatin and plasminogen binding to endothelial cells by 68 and 62%, respectively, supporting our in vitro studies that annexin II is a receptor for angiostatin. Angiostatin-binding protein/annexin II specifically expressed in endothelial cells but not in fibroblasts suggests its EC-specific function. Epsilon-aminocaproic acid, a lys analogue, effectively blocks angiostatin and annexin II interaction, indicating that the lysine-binding domain of AS is required for binding to annexin II. These results suggest that the antiangiogenic action of angiostatin may be mediated via interaction with annexin II. Identification of annexin II as a receptor for angiostatin provides further evidence that clotting and fibrinolytic pathways are directly involved in the angiogenic process.