SPARC-null mice exhibit accelerated cutaneous wound closure.

Expression of SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40), an extracellular matrix (ECM) associated protein, is coincident with matrix remodeling. To further identify the functions of SPARC in vivo, we have made excisional wounds on the dorsa of SPARC-null and wild-type mice and monitored closure over time. A significant decrease in the size of the SPARC-null wounds, in comparison to that of wild-type, was observed at Day 4 and was maximal at Day 7. Although substantial differences in the percentage of proliferating cells were not apparent in SPARC-null relative to wild-type wounds, primary cultures of SPARC-null dermal fibroblasts displayed accelerated migration, relative to wild-type fibroblasts, in wound assays in vitro. Although the expression of collagen I mRNA in wounds, as measured by in situ hybridization (ISH), was not significantly different in SPARC-null vs wild-type mice, the collagen content of unwounded skin appeared to be substantially lower in the SPARC-null animals. By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of wild-type skin. Moreover, we found an inverse correlation between the efficiency of collagen gel contraction by dermal fibroblasts and the concentration of collagen within the gel itself. We propose that the accelerated wound closure seen in SPARC-null dermis results from its decreased collagen content, a condition contributing to enhanced contractibility.     

Enhanced growth of pancreatic tumors in SPARC-null mice is associated with decreased deposition of extracellular matrix and reduced tumor cell apoptosis

SPARC, a matricellular glycoprotein, modulates cellular interaction with the extracellular matrix (ECM). Tumor growth and metastasis occur in the context of the ECM, the levels and deposition of which are controlled in part by SPARC. Tumor-derived SPARC is reported to stimulate or retard tumor progression depending on the tumor type, whereas the function of host-derived SPARC in tumorigenesis has not been explored fully. To evaluate the function of endogenous SPARC, we have examined the growth of pancreatic tumors in SPARC-null (SP(-/-)) mice and their wild-type (SP(+/+)) counterparts. Mouse pancreatic adenocarcinoma cells injected s.c. grew significantly faster in SP(-/-) mice than cells injected into SP(+/+) animals, with mean tumor weights at sacrifice of 0.415 +/- 0.08 and 0.086 +/- 0.03 g (P < 0.01), respectively. Lack of endogenous SPARC resulted in decreased collagen deposition and fiber formation, alterations in the distribution of tumor-infiltrating macrophages, and decreased tumor cell apoptosis. There was no difference in microvessel density of tumors from SP(-/-) or SP(+/+) mice. However, tumors grown in SP(-/-) had a lower percentage of blood vessels that expressed smooth muscle alpha-actin, a marker of pericytes. These data reflect the importance of ECM deposition in regulating tumor growth and demonstrate that host-derived SPARC is a critical factor in the response of host tissue to tumorigenesis.     

Mammary tumorigenesis in ApcMin/+ mice is enhanced by X irradiation with a characteristic age dependence

The Apc(Min/+) (Min) mouse is genetically predisposed to both intestinal and mammary tumorigenesis. We investigated age-related changes in the susceptibility of mice (before, during and after puberty) to radiation-induced mammary tumorigenesis using this model. Female Min and wild-type mice having the C57BL/6J background were irradiated with 2 Gy of X rays at 2, 5, 7 and 10 weeks and killed humanely at 18 weeks of age. Min mice irradiated at 7-10 weeks of age (after puberty) developed mammary tumors with squamous metaplasia, whereas their wild-type littermates did not. Interestingly, irradiation of Min mice at 2-5 weeks (before and during puberty, respectively) did not induce mammary tumors but rather cystic nodules with metaplasia. The mammary tumors exhibited increased nuclear beta-catenin protein and loss of the wild-type Apc allele. Our results show that susceptibility to radiation-induced mammary tumorigenesis increases after puberty in Min mice, suggesting that the tumorigenic effect of ionizing radiation targets the lobular-alveolar progenitor cells, which increase in number with age and are controlled by beta-catenin signaling.     

Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.     

Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.     

Genetic heterogeneity of angiogenesis in mice.

Many diseases, including cancer, are dependent on the growth of new blood vessels, a process known as angiogenesis. Differences in an individual's ability to grow new blood vessels may influence the rate of progression of these diseases. Here we show that different strains of inbred mice have an approximately 10-fold range of response to growth factor-stimulated angiogenesis in the corneal micropocket assay. The in vitro migratory activity of endothelial cells from aortic rings of selected strains correlated with the in vivo responsiveness. Further, a differential sensitivity to angiogenesis inhibitors was seen between strains, with one strain demonstrating resistance to both TNP-470 and thalidomide. These results suggest the presence of genetic factors that control individual angiogenic potential.     

Electroencephalographic changes with age in male mice.

Electroencephalographic (EEG) changes, as measured by the awake state, slow-wave sleep (SWS), rapid-eye movement (REM) patterns and ratio of REM/total sleep, were recorded in aging male mice of DBA/2J and C57BL/6J strains. Results indicate that there is a significant increase in the awake state accompanied by significant decrease in SWS with advancing age for both strains, although these changes appear more pronounced in DBA/2J mice than C57BL/6J mice. Of considerable significance is the finding that REM sleep is absent in mice of DBA/2J strain at 23.5 months of age. Based on these findings, the conclusion was reached that strain DBA/2J ages significantly faster than C57BL/6J. The difference in aging between the two strains emphasizes the need for additional studies dealing with genetic aspects of aging.     

Enhanced tumor susceptibility of immunocompetent mice infected with lymphocytic choriomeningitis virus

Summary Mice infected i.v. with high doses of lymphocytic choriomeningitis virus (LCMV; 10⁵–10⁶ plaqueforming units) 8–10 days prior to challenge with the methylcholanthrene-induced fibrosarcoma tumor cell line MC57G or the melanoma cell line B16 tumor cells showed an enhanced tumor susceptibility with respect to both growth kinetics of the tumor and the minimal dose necessary for tumor take. After transient initial growth, MC57G tumor cells were all rejected by uninfected C57BL/6 mice by day 14. Mice preinfected i.v. with LCMV 3 weeks before or at the time of tumor challenge, but not those infected 2 months before or 7 days after, showed increasing tumor growth, the tumor take being 100% for 10⁶, 50% for 10⁵ and 37% for 10⁴ MC57G tumor cells injected into the footpad compared with resistance to 10⁶ cells in normal mice. B16 melanoma cells also grew more rapidly in LCMV-preinfected mice and by day 40 tumors were established with about 100 times fewer cells, i.e. about 10³ compared with 3×10⁴–3×10⁵ for uninfected mice. Analysis of the growth of tumor cells in normal and in LCMV-carrier mice revealed that the latter mice were not more susceptible to LCMV-infected than to uninfected MC57G. Since LCMV-carrier mice fail to mount LCMV-specific T cell responses, these results suggest that anti-LCMV-specific T cells may be responsible for acquired immunodeficiency hampering immune surveillance against the tumors studied.Supported by grants from the Swiss National Science Foundation 3.259–0.87 and the Kanton of Zürich     

Enhanced arteriogenesis in mice overexpressing erythropoietin

After permanent occlusion of the femoral artery, the survival of ischemic limb tissue depends on collateral artery growth (arteriogenesis). In previous work, we have shown that shear stress triggers arteriogenesis. To test whether increased shear stress results in enhanced arteriogenesis, we compared arteriogenesis in transgenic mice overexpressing erythropoietin (EPO), which possessed increased blood viscosity through the higher hematocrit (thereby providing increased shear stress), with wild-type mice. The right femoral artery was occluded proximal to the origin of the arteria poplitea. Distal blood flow was assessed by laser Doppler imaging, and the growth and remodeling of collateral arteries was examined by light and electron microscopy and morphometry. After occlusion of the femoral artery, EPO mice demonstrated enhanced arteriogenesis: their collateral arteries developed a 1.7-fold diameter and a 2-fold wall thickness compared with wild-type. However, the blood flow recovery in EPO mice was markedly retarded. Structural remodeling and growth of collateral arteries was markedly enhanced in EPO mice, presumably as a result of increased blood viscosity and shear stress.     

Protease-mediated enhancement of lymphocyte-induced angiogenesis in X-ray irradiated mice

Angiogenesis was induced in mice by intradermal injection of semi-syngeneic splenocytes, and after three days the number of newly formed blood vessels at the injection site was counted. When recipients were total-body irradiated with 700 R 2 hours before the lymphocyte injection, the angiogenesis was significantly higher than in non-irradiated mice. The angiogenesis enhancement was of a systemic (not local) character as revealed in experiments with shielding of irradiated animals. This enhancement was not due to X-ray dependent immunosuppression, as shown in experiments with non-irradiated, pharmacologically immunosuppressed mice. Decreased angiogenesis was observed in irradiated mice after treatment with cortisone acetate, aprotinin, and EACA. The results suggest that proteases might be involved in mediating the angiogenesis enhancement after X-irradiation.     

Enhanced resistance of restraint-stressed mice to sepsis

Sepsis remains a major health concern across the world. The effects of stress on host resistance to sepsis are still not very clear. To explore the effects of chronic stress on sepsis(') we examined the impact of restraint stress on the resistance of mice to sepsis. Interestingly, it was found that restraint stress enhanced the antisepsis resistance of mice and the concentrations of the proinflammatory cytokines IL-1, IL-6, IL-12, and TNF-alpha in the blood of stressed mice were dramatically reduced post Escherichia coli infection or LPS treatment as compared with that of controls (p < 0.05). In addition, the mRNA expressions of glucocorticoid-induced leucine zipper (GILZ) were up-regulated in the spleen and peritoneal macrophages of mice receiving restraint stress or dexamethasone treatment. These results demonstrate that restraint stress enhances the resistance of mice to sepsis, supporting corticotherapy for sepsis and proposing restraint-stressed mouse as an animal model to elucidate mechanisms of stress-associated, antisepsis resistance.     

Enhanced autophagy blocks angiogenesis via degradation of gastrin-releasing peptide in neuroblastoma cells

Neuroblastoma is characterized by florid vascularization leading to rapid tumor dissemination to distant organs; angiogenesis contributes to tumor progression and poor clinical outcomes. We have previously demonstrated an increased expression of gastrin-releasing peptide (GRP) and its receptor, GRPR, in neuroblastoma and that GRP activates the PI3K-AKT pathway as a proangiogenic factor during tumor progression. Interestingly, AKT activation phosphorylates MTOR, a critical negative regulator of autophagy, a cellular process involved in the degradation of key proteins. We hypothesize that inhibition of GRPR enhances autophagy-mediated degradation of GRP and subsequent inhibition of angiogenesis in neuroblastoma. Here, we demonstrated a novel phenomenon where targeting GRPR using shRNA or a specific antagonist, RC-3095, decreased GRP secretion by neuroblastoma cells and tubule formation by endothelial cells in vitro. Furthermore, shGRPR or RC-3095 treatment enhanced expression of proautophagic proteins in human neuroblastoma cell lines, BE(2)-C, and BE(2)-M17. Interestingly, rapamycin, an inhibitor of MTOR, enhanced the expression of the autophagosomal marker LC3-II and GRP was localized within LC3-II-marked autophagosomes in vitro as well as in vivo, indicating autophagy-mediated degradation of GRP. Moreover, overexpression of ATG5 or BECN1 attenuated GRP secretion and tubule formation, whereas opposite effects were observed with siRNA silencing of ATG5 and BECN1. Our data supported the role of autophagy in the degradation of GRP and subsequent inhibition of angiogenesis. Therefore, activation of autophagy may lead to novel antivascular therapeutic strategies in the treatment of highly vascular neuroblastomas.     

SARI prevents ocular angiogenesis and inflammation in mice

SARI (Suppressor of AP-1, regulated by IFN-β) is known to play an important role in some systemic disease processes such an inflammatory conditions and cancer. We hypothesize that SARI may also play a role in ocular diseases involving inflammation and neovascularization. To explore our hypothesis, further, we investigated an endotoxin-induced uveitis (EIU) and experimental argon laser-induced choroidal neovascularization (CNV) model in SARI wild-type (SARI^WT) and SARI-deficient (SARI^−/−) mice. Through imaging, morphological and immunohistochemical (IHC) studies, we found that SARI deficiency exacerbated the growth of CNV. More VEGF-positive cells were presented in the retina of SARI^−/− mice with CNV. Compared to SARI^WT mice, more inflammatory cells infiltrated the ocular anterior segment and posterior segments in SARI^−/− mice with EIU. Collectively, the results point to a potential dual functional role of SARI in inflammatory ocular diseases, suggesting that SARI could be a potential therapy target for ocular inflammation and neovascularization.     

Enhanced cellular response in mice treated with a Brucella antigen-liposome mixture

Abstract The capacity of liposomes constituted by dycetyl-phosphate (0.009 mM), cholesterol (0.017 nM), lecithin (0.003 mM), and myristic (0.1 mM), stearic (0.1 mM), or oleic acid (0.1 mM) to modify the lymphocyte response to Brucella melitensis antigens in mice was studied. Mice treated with antigens mixed with liposomes containing myristic, stearic or oleic acid had higher antibody titres than mice given antigen suspended in a saline solution. Liposomes alone, without Brucella antigens, resulted in increased ³H-thymidine incorporation by lymphocytes both in vivo and in vitro. The addition of polyclonal activators (LPS and ConA) caused a further increase of ³H-thymidine uptake. Moreover, spleen lymphocytes from mice inoculated with Brucella antigens mixed with the liposomes had a significantly lower population of B lymphocytes (10%), and a notable increase in the T_c lymphocytes (20%). Autoradiography of sections of popliteal ganglia of treated mice showed that the radioactivity was concentrated mainly in the membrane structures of the cell.     

Enhanced endothelial nitric oxide-synthase activity in mice infected with Trypanosoma brucei

Infection of humans with Trypanosoma brucei causes sleeping sickness, which is invariably fatal if left untreated. The course of infection is characterised, among others, by multiple organ damage including cardiovascular dysfunctions such as hypotension and breakdown of the blood-brain barrier. The latter eventually leads to the parasite invasion into central nervous system and ultimately to the death of the patient. Nitric oxide (NO) synthesised from L-arginine via endothelial NO-synthase (eNOS) is involved in the control of vascular tone and permeability. The present study explores the effect of T. brucei infection on the endothelium-dependent in vitro vasomotor response of isolated mouse aortas. Aorta rings were suspended in organ chambers for isometric tension recording. The endothelium-dependent NO-mediated relaxation in response to acetylcholine (10(-9) to 10(-5) M) was markedly enhanced in the infected mice compared to controls (P<0.05), whereas the endothelium-independent vasodilation to an exogenous NO-donor, sodium nitroprusside, was comparable in both groups. Norepinephrine-stimulated contraction was also comparable in the absence or presence of the NO-synthase inhibitor N(omega)-Nitro-L-arginine methyl ester (L-NAME; 10(-4)M) in both groups. The enhanced endothelium-dependent relaxation in the infected mice correlated well with a 3.5-fold increase in eNOS protein level in these aortas as compared to those of control mice (P=0.05). Thus, T. brucei infection enhances eNOS protein expression in the endothelium, causing a pronounced vasodilation. Overproduction of NO in trypanosomiasis may be involved in the observed generalised hypotension and in an increased vascular permeability that facilitates T. brucei invasion into surrounding tissues and its penetration into the central nervous system in later phases of infection.     

Enhanced oral tolerance in transgenic mice with hepatocyte secretion of IL-10

Several cytokines derived from Th3 and Tr1 cells, including IL-10, are believed to regulate oral tolerance, but direct evidence is lacking. We have explored the potential role of IL-10 by generating transgenic (TG) mice with sustained hepatocyte-specific expression of rat IL-10. TG mice expressed rat IL-10 downstream of a transthyretin promoter, which led to serum levels that were increased 10- to 100-fold compared with normal animals. Animals were orally administered 1 mg of whole OVA for 5 consecutive days, with control animals receiving PBS. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. On day 8, all mice were immunized with two injections of OVA, and then analyzed on day 18. T cell proliferation responses were reduced by 65.8 +/- 14.3% after feeding of OVA in rIL-10 TG animals, compared with 39.4 +/- 15.6% in the non-TG mice (p = 0.02). Anti-OVA titers were expressed as fold increase over naive non-TG mice. After feeding, titers decreased by approximately 33% (from 3- to 2-fold) in TG animals and, to a lesser extent, in non-TG animals. IFN-gamma secretion by cultured popliteal lymphocytes decreased in TG animals by 83% after feeding and by 69% in non-TG animals. IL-4 secretion increased 4-fold in TG-fed mice, but did not significantly change in non-TG OVA-fed animals. In contrast to hepatic TG expression of rIL-10, systemic administration of rIL-10 had only a modest effect on tolerance. IL-10, when transgenically expressed in the liver enhances mucosal tolerance to an oral Ag.     

Decline in the production of interleukin-3 with age in mice

Previously, we and others have found that the ability to produce interleukin-1 (IL-1) and interleukin-2 (IL-2) declines with age in mice. The purpose of this study was to determine the influence of age on the capacity of mice to produce interleukin-3 (IL-3). Splenic cells (5 X 10(6)/ml) from young (3-4 months) and old (24-32 months) C57BL/6 mice were first assessed for their IL-3-producing capacities in response to varying doses of concanavalin A (Con A; 2-20 micrograms/ml) in a time-dependent manner. The results showed that the production of IL-3 by both young and old C57BL/6 mice was maximal on Days 3 and 4 in response to 20 micrograms/ml of Con A, and that of IL-2 was minimal (activity was less than 0.1 unit) on Day 4. Consequently, Day 4, was selected to assess the effect of age on IL-3 production by splenic cells. The results showed a twofold reduction in IL-3 production with age (P less than 0.05). Young-old splenic cell mixture experiments at ratios of 1:0, 3:1, 1:1, 1:3, and 0:1 indicated that the decrease in IL-3 production with age was not due to an increase in suppressor cell activity. Experiments based on mixtures of nylon wool-enriched splenic T-cell and adherent cells and on anti-MAC-1 plus complement-treated spleen cells indicated that (a) adherent cells are not required for T-cell production of IL-3, unlike IL-2 production, and (b) the decrease in IL-3 production with age is due solely to alteration in IL-3-producing T cells. Finally, a strong correlation was demonstrated between the production of IL-2 and IL-3 by spleen cells of individual young and old mice (r = 0.92, P less than 0.01). That production of both IL-2 and IL-3 is affected in a similar manner by age would suggest that a single class of helper T cells may be responsible for production of both lymphokines.     

Enhanced plutonium absorption in iron-deficient mice.

The total body burden of plutonium 24 or 96 hr following a single gastric intubation was approximately fourfold greater in iron-deficient than in iron-replete mice. There was also a more rapid translocation of plutonium from soft tissues to bone in the iron-deficient mice by 96 hr after gavage. In the iron-replete group only liver concentrations of plutonium increased during the corresponding time period.     

Inhibition of inflammatory angiogenesis by distant subcutaneous tumor in mice

We investigated angiogenesis, inflammatory cells accumulation and endogenous production of cytokines in sponge implants of tumor-bearing mice. Seven days after inoculation of Ehrlich tumor cells (2.5 x 10(6)), sponge discs were implanted subcutaneously in the dorsa of mice to induce the formation of fibrovascular tissue. The implants of tumor-bearing and non tumor-bearing animals were assessed for neovascularization and leukocyte accumulation, together with levels of relevant cytokines, vascular endothelial growth factor VEGF), tumor necrosis factor alpha (TNF-alpha), CXCL1-3/KC and CCL2/JE. In the implants of tumor-bearing animals angiogenesis (assessed by hemoglobin content and VEGF levels in the implants) and leukocyte accumulation (assessed by myeloperoxidase -MPO- and N- acetylglucosaminidase-NAG-enzyme activities) were all significantly less than those in the implants of non tumor-bearing animals. Although the chemokine CXCL1-3/KC was lower in the implants of tumor-bearing animals, the chemokine CCL2/JE was increased in this group. The production of TNF-alpha in the implants was not modified by the presence of the subcutaneous tumor. The combination of the methodologies used in this study has provided a novel approach to investigate the interaction between two distinct proliferating tissues that share common features (angiogenesis, cell recruitment, inflammation) and has shown that the predominant inhibitory effect of a tumor mass over repair process is associated with altered cytokine production.