Disruption of the TIMP-1 gene product is associated with accelerated endometrial gland formation during early postnatal uterine development

Postnatal uterine development is marked by periods of tissue remodeling. The objective of the present study was to examine the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a regulator of tissue remodeling events, during postnatal uterine development and to assess the phenotypic consequences of disruption of the TIMP-1 gene product during this time period. To accomplish this goal, wild-type and TIMP-1 null mice were sacrificed at Postnatal Days (PNDs) 5, 10, 15, 20, and 25 and uterine morphology, TIMP expression and matrix metalloproteinase (MMP) activity were assessed. In wild-type mice, TIMP-1 mRNA steady-state levels were highest at PND 5, after which expression decreased. TIMP-2 and TIMP-3 expression in wild-type mice showed no significant changes from PND 5 to 25. In TIMP-1 null mice, TIMP-2 and TIMP-3 expression patterns were similar to those in wild-type counterparts with the exception that, at PND 10, TIMP-2 and TIMP-3 expression was significantly lower in the null mice. Endometrial gland number and uterine histology were similar between genotypes at PNDs 5 and 10, but at PNDs 15 and 20, endometrial glands were more abundant in TIMP-1 null mice. Associated with the increased gland density in the null mice was an increase in total MMP activity above the levels expressed in wild-type mice. In summary, disruption of the TIMP-1 gene product is associated with reduced TIMP-2 and TIMP-3 steady-state mRNA levels, elevated MMP activity, and accelerated endometrial gland formation. We conclude that, during early postnatal uterine development, TIMP-1 may be critical for proper endometrial gland development.     

Plasminogen activator inhibitor 1 (PAI-1) 1334G/A genetic polymorphism in colorectal cancer

Plasminogen activator inhibitor 1 (PAI-1) content in colorectal cancer tissue extracts may be of strong prognostic value: high levels of PAI-1 in tumours predict poor prognosis. The gene encoding PAI-1 is highly polymorphic and PAI-1 gene variability could contribute to the level of PAI-1 biosynthesis. In the present work the distribution of genotypes and frequency of alleles of the 1334G/A polymorphism in 92 subjects with colorectal cancer in samples of cancer tissue and distant mucosa samples as well as in blood were investigated. Blood samples age matched healthy individuals (n = 110) served as control. The 1334G/A polymorphism was determined by PCR amplification using allele specific primers. No differences in the genotype distributions and allele frequencies between blood, distant mucosa samples and cancer tissue were detected. However, the distribution of the genotypes of the 1334G/A polymorphism in patients differed significantly (P <0.05) from those predicted by the Hardy-Weinberg equilibrium. There were significant differences in the frequencies of alleles between the colorectal cancer subjects and controls (P <0.05). The results support the hypothesis that the 1334G/A polymorphism may be associated with the incidence of colorectal cancer.     

ANG II increases TIMP-1 expression in rat aortic smooth muscle cells in vivo

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tissue remodeling processes. TIMP-1 is the main native inhibitor of MMPs and it contributes to the development of tissue fibrosis. It is known that ANG II plays a fundamental role in vascular remodeling. In this study, we investigated whether ANG II modulates TIMP-1 expression in rat aortic smooth muscle cells. In vitro, ANG II induces TIMP-1 mRNA expression in a dose-dependent manner. The maximal increase in TIMP-1 expression was present after 3 h of ANG II stimulation. The ANG II increase in TIMP-1 expression was mediated by the ANG type 1 receptors because it was blocked by losartan. The increase in TIMP-1 expression was present after the first ANG II treatment, whereas repeated treatments (3 and 5 times) did not modify TIMP-1 expression. In vivo, exogenous ANG II was administered to Sprague-Dawley rats (200 ng. kg(-1). min(-1) sc) for 6 and 25 days. Control rats received physiological saline. After treatment, systolic blood pressure was significantly higher (P < 0.01), whereas plasma renin activity was suppressed (P < 0.01), in ANG II-treated rats. ANG II increased TIMP-1 expression in the aorta of ANG II-treated rats both at the mRNA (P < 0.05) and protein levels as evaluated by Western blotting (P < 0.05) and/or immunohistochemistry. Neither histological modifications at the vascular wall nor differences in collagen content in the tunica media were present in both the ANG II- and saline-treated groups. Our data demonstrate that ANG II increases TIMP-1 expression in rat aortic smooth muscle cells. In vivo, both short- and long-term chronic ANG II treatments increase TIMP-1 expression in the rat aorta. TIMP-1 induction by ANG II in aortic smooth muscle cells occurs in the absence of histological changes at the vascular wall.     

Disruption of the tissue inhibitor of metalloproteinase-1 gene results in altered reproductive cyclicity and uterine morphology in reproductive-age female mice

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a multifunctional protein expressed in the uterus of essentially all species, yet the function of this protein is uncertain. To assess the role of TIMP-1 in the uterine events that occur during the murine estrous cycle, mature female TIMP-1 wild-type and null mice were monitored for reproductive cyclicity. Mice were sacrificed in each stage of the estrous cycle, and peripheral blood was collected and assayed for serum estradiol and progesterone content by RIA. Uterine morphology and TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA expression were also examined between genotypes in each stage of the estrous cycle. Disruption of the TIMP-1 gene product was associated with an altered reproductive cycle characterized by a significant decrease in the length of the estrus period in the null mice. Also during the period of estrus, null mice expressed significantly lower levels of uterine TIMP-3 mRNA expression, altered uterine morphology, significantly higher serum estradiol levels, and significantly lower serum progesterone levels compared to their wild-type counterparts. It is concluded from this study that TIMP-1 has a multifaceted role in regulating the murine reproductive cycle, and this control appears to be at the level of both the uterus and the ovary.     

Maternal dexamethasone treatment at midgestation reduces nephron number and alters renal gene expression in the fetal spiny mouse

We investigated the effects of maternal glucocorticoid exposure in the spiny mouse, a precocial species with a relatively long gestation, few offspring, and in which nephrogenesis is complete before birth. We hypothesized that exposure of the fetus to glucocorticoids before the formation of glomeruli would result in adult hypertensive offspring with fewer nephrons. Furthermore, we hypothesized that this nephron deficit would result from changes in expression of genes involved in branching morphogenesis. Osmotic pumps implanted in pregnant spiny mice at midgestation (day 20) delivered dexamethasone (dex; 125 microg/kg) or saline for 60 h. Females were killed at day 23 of gestation and kidneys were frozen for real-time PCR analysis or allowed to deliver their offspring. At 20 wk of age, blood pressure was measured in the offspring for 1 wk before nephron number was determined using unbiased stereology. Males and females exposed to dex had significantly fewer nephrons (male: saline: 7,870 +/- 27, dex: 6,878 +/- 173; female: saline: 7,526 +/- 62, dex: 5,886 +/- 382; P < 0.001) compared with controls. Dex had no effect on basal blood pressure. Fetal kidneys collected at day 23 of gestation from dex-exposed mothers showed increased mRNA expression of BMP4 (P < 0.05), TGF-beta(1) (P < 0.05), genes known to inhibit branching morphogenesis and gremlin (P < 0.01), an antagonist of BMP4, compared with saline controls. This study shows for the first time an upregulation of branching morphogenic genes in the fetal kidney in a model of excess maternal glucocorticoids that leads to a nephron deficit in the adult. This study also provides evidence that a reduced nephron number does not necessarily lead to development of hypertension.     

Localization of preovulatory expression of plasminogen activator inhibitor type-1 and tissue inhibitor of metalloproteinase type-1 mRNAs in the rat ovary.

Proteinases and their inhibitors control follicular connective tissue remodeling associated with follicular rupture. We examined the regulation and cellular localization of plasminogen activator inhibitor type-1 (PAI-1) and tissue inhibitor of metalloproteinase type-1 (TIMP-1) mRNAs by in situ hybridization. [35S]UTP-labeled RNA probes were hybridized to ovarian sections of eCG-primed immature rats treated with hCG. Before hCG stimulation of ovulation, very low expression of PAI-1 mRNA was observed in theca cells. After hCG administration, expression of PAI-1 mRNA was increased in theca cells of most antral follicles, whereas expression in granulosa cells was limited to preovulatory follicles and only to areas where the basal membrane was dissociated. Before hCG treatment, low expression of TIMP-1 mRNA was observed in theca cells, but not in granulosa cells. After hCG treatment, TIMP-1 mRNA was greatly stimulated in theca cells irrespective of follicle size, while the expression in granulosa cells was limited to large antral follicles. The present study demonstrates cell-specific expression of PAI-1 and TIMP-1 mRNAs in the LH/hCG-stimulated ovary, thus confirming the localized control of preovulatory proteolysis by coexpression of both enzymes and their respective inhibitors.     

Tumor tissue concentrations of the proteinase inhibitors tissue inhibitor of metalloproteinases-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) are complementary in determining prognosis in primary breast cancer

The purpose of this study was to investigate the association between tumor tissue levels of total tissue inhibitor of metalloproteinases-1 (TIMP-1) and prognosis in patients with primary breast cancer and to analyze whether measurement of TIMP-1 in tumor extracts added prognostic information to that obtained from measurements of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 (PAI-1). An established sandwich enzyme-linked immunosorbent assay was thoroughly validated for the measurement of total TIMP-1 in tumor tissue extracts and used to determine levels of total TIMP-1 in 341 detergent-extracted tumor tissue samples from patients with primary breast cancer. The median age of the patients was 56 years (range, 29-75 years), and 164 were lymph node-negative, and 177 were lymph node-positive. The median follow-up time of the patients was 8.5 years (range, 7.3-11.3 years), and during follow-up 153 patients experienced recurrence of disease, and 136 patients died. In univariate survival analysis, we found a significant association between tumor tissue TIMP-1 level and both shorter recurrence-free survival (p = 0.0004) and shorter overall survival (p = 0.03). In multivariate survival analysis, higher tumor tissue TIMP-1 levels significantly and independently predicted shorter recurrence-free survival (p < 0.05, hazard ratios >1, comparing quartiles II-IV with I). In addition, we found that measurement of TIMP-1 levels added prognostic information to that obtained from measurement of PAI-1. In conclusion, high levels of TIMP-1 in tumor tissue extracts are significantly associated with a poor prognosis in patients with primary breast cancer. Furthermore TIMP-1 adds prognostic information to that obtained from PAI-1. However, further validation in independent data sets is needed.     

A possible maternal effect in the abnormal hyporesponsiveness to specific alloantigens in offspring born to neonatally tolerant fathers

Newborn CBA mice were inoculated i.p. with 1 X 10(8) adult (CBA X A)F1 lymphoid cells with 5 X 10(7) F1 hybrid lymphoid cells thereafter inoculated i.v. at 14-day intervals. The mice were subsequently grafted with A/J tail skin at 24 days of age. At 8 wk of age, individual tolerant males with intact skin grafts were each mated with similar tolerant skin-grafted females or with 8-wk-old control normal CBA females. Approximately 0.4 ml of peripheral blood from 7-wk-old progeny born to these crosses, along with the blood from age-matched progeny born to normal CBA incrosses, were used for in vitro mixed leukocyte cultures (MLC). All progeny received skin grafts of (CBA X A)F1 tail skin at 7 1/2 wk of age. In this study, we show i) that MLC hyporesponsiveness to histocompatible cells is an acquired phenotype expressed by lymphoid cells of the progeny of tolerant male mice, and ii) that these progeny also show delayed rejection of (CBA X A)F1 skin grafts. The continued breeding of the first generation animals established that a similar transmission of hyporesponsiveness can be observed in the second generation progeny. When putatively normal females, however, which had borne several litters after mating with neonatally tolerant males, were mated with "normal" (nontolerant) males; their offspring also exhibited a specific hyporesponsiveness in MLC to the initial tolerizing allodeterminants. This suggests that there may ultimately be a maternally derived origin for the transmission of characters from tolerant males.     

PSGL-1基因敲除小鼠血中MIP-1γ和TNF-α的表达

目的 研究PSGL-1 缺失对遗传基因工程小鼠外周血血常规的影响,并检测外周血中炎症因子IGFBP-6、TNF-α和MIP-1γmRNA 表达水平.方法 用血常规检测方法检测正常C57/BL/6 小鼠和PSGL-1 基因缺失的基因工程小鼠（PSGL-1 -/-小鼠）的外周血中血常规的差异;其次,提取两种鼠的血液的mRNA,逆转录为cDNA,采用real-time PCR 方法,检测C57 小鼠与PSGL-1 -/-小鼠外周血中炎症因子TNF-α和MIP-1γmRNA 的差异.结果 与对照组C57 小鼠相比,PSGL-1 -/-基因工程小鼠在12 周龄时外周血中中性粒细胞（倡P ＜0.05）、淋巴细胞（倡倡P ＜0.01）和白细胞细胞（倡倡倡P ＜0.001）总数显著增加炎症因子TNF-α以及MIP-1γ表达增加（P ＜0.05）.结论 PSGL-1 的缺失改变了小鼠细胞因子并影响了外周血的血细胞组成,MIP-1γ和TNF-α炎症因子上调,有可能影响了小鼠的免疫功能.     

Plasminogen activator inhibitor-1 (PAI-1) gene 4G/5G promoter polymorphism is not associated with breast cancer

The antigen content of plasminogen activator inhibitor-1 (PAI-1) in primary breast cancer tissue extracts may be of strong prognostic value: high levels of PAI-1 in tumors predict poor prognosis for patients. The gene encoding PAI-1 is highly polymorphic and an insertion (5G)/deletion (4G) polymorphism in the PAI-1 gene promoter (the 4G/5G polymorphism), may have functional significance in PAI-1 expression. In the present work the distribution of genotypes and frequency of alleles of the 4G/5G polymorphism in subjects with breast cancer were investigated. Tumor tissues were obtained from 100 postmenopausal women with node-negative and node-positive ductal breast carcinoma with uniform tumor size. Blood samples from age matched healthy women served as control. The 4G/5G polymorphism was determined by PCR amplification using the allele specific primers. The distribution of the genotypes of the 4G/5G polymorphism in both control and patients did not differ significantly (P > 0.05) from those predicted by the Hardy-Weinberg distribution. There were no differences in the genotype distributions and allele frequencies between node-positive and node-negative patients. The 4G/5G polymorphism may not be linked with elevated level of PAI-1 observed in breast cancer and therefore may not be associated with appearance and/or progression of breast cancer.     

Expression of matrix metalloproteinases and their tissue inhibitors in the serum and cerebrospinal fluid of patients with HIV-1 infection and syphilis or neurosyphilis

The potential mechanisms for altered matrix metalloproteinase (MMP) or tissue inhibitors of matrix metalloproteinase (TIMP) function in patients with syphilis and HIV-1 co-infection (HIV-S) was unclear. To determine the expression of MMP-2, 9 and TIMP-1, 2, 4 in the serum and cerebrospinal fluid (CSF) of HIV-S patients, a total of 20 HIV-S patients and 8 controls were enrolled in a HIV-1 clinical cohort for diagnosis of neurosyphilis in Taiwan. Serum and CSF concentrations of MMP-2, 9, and TIMP-1, 2, 4 were determined by ELISA. Gelatin zymography was used to detect the expression of MMP-2 and MMP-9 in the CSF. Neurosyphilis was defined as a CSF white blood cell count ≥ 20 cells/μL or a reactive CSF Venereal Disease Research Laboratory (VDRL). All the patients with HIV-S were males. Most (85%) had sex with men (MSM) and serum rapid plasma reagin (RPR) titers of ≥ 1:32. The median age was 35 years (IQR 30-43). The median CD4 T cell counts at the time of the diagnosis of syphilis were 270 cells/μL (IQR 96-484). Ten patients (50%) had neurosyphilis based on a reactive CSF VDRL test (n=8) or increased CSF white cell counts ≥ 20/μL (n=2). The concentrations of CSF MMP-9, TIMP-1, and TIMP-2 were significantly higher in patients with HIV-S than the controls (P<0.05). The CSF TIMP-4 concentrations were significantly lower in those with HIV-S (452 pg/ml) than controls (3101 pg/ml), P<0001. There were no significant differences in serum concentrations between the groups. The only finding that distinguished HIV-1 patients with from those without neurosyphilis is a significant higher expression of CSF MMP-9. In conclusion, the MMP/TIMP system was found to be dysregulated in patients with HIV-S regardless of whether they met the laboratory definition of neurosyphilis. The CSF level of MMP-9 was the only measure that distinguished those with or without neurosyphilis.     

Differential expression of plasminogen activator inhibitor-1, tumor necrosis factor-alpha,TNF-alpha converting enzyme and ADAMTS family members in murine fat territories

Our objective was to investigate expression of A disintegrin and metalloproteinase (ADAM) and ADAM proteins with a thrombospondin (TS) motif (ADAMTS) family members in adipose tissue of lean and obese mice. Five-week-old male mice were kept on standard chow (SFD) or on high fat diet (HFD) for 15 weeks, and subcutaneous (SC) and gonadal (GON) adipose tissue, as well as mature adipocytes and stromal-vascular (S-V) cells were harvested. mRNA levels of plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-alpha (TNF-alpha), ADAM-17 (TACE or TNF-alpha converting enzyme), ADAMTS-1 and ADAMTS-8 were quantified in isolated adipose tissues and cell fractions, and during differentiation of murine preadipocytes. The HFD resulted in a significantly enhanced weight of isolated SC and GON fat pads, and in enhanced blood levels of glucose, cholesterol and PAI-1. ADAM-17, TNF-alpha, PAI-1, ADAMTS-1 and ADAMTS-8 mRNA were detected in both SC and GON adipose tissue of lean mice (SFD). In SC adipose tissue of obese mice (HFD), the expression of ADAM-17 and PAI-1 was enhanced and that of ADAMTS-1 reduced, whereas in GON adipose tissue expression of TNF-alpha was enhanced and that of ADAMTS-8 reduced. In lean and obese mice, expression of ADAM-17, ADAMTS-1 and ADAMTS-8 was higher in the S-V cell fraction than in mature adipocytes. During differentiation of murine 3T3-F442A preadipocytes, expression of ADAM-17 and ADAMTS-1 remained virtually unaltered, whereas that of ADAMTS-8 decreased as adipocytes matured. Several ADAM and ADAMTS family members are expressed in adipose tissue and during differentiation of preadipocytes. Modulation of their expression upon development of obesity is adipose tissue-dependent.     

Regulatory role of NADPH oxidase in glycated LDL-induced upregulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts and diabetic mice

Cardiovascular disease is the predominant cause of death in diabetic patients. Fibroblasts are one of the major types of cells in the heart or vascular wall. Increased levels of glycated low-density lipoprotein (glyLDL) were detected in diabetic patients. Previous studies in our group demonstrated that oxidized LDL increased the amounts of NADPH oxidase (NOX), plasminogen activator inhibitor-1 (PAI-1), and heat shock factor-1 (HSF1) in fibroblasts. This study examined the expression of NOX, PAI-1, and HSF1 in glyLDL-treated wild-type or HSF1-deficient mouse embryo fibroblasts (MEFs) and in leptin receptor-knockout (db/db) diabetic mice. Treatment with physiologically relevant levels of glyLDL increased superoxide and H₂O₂ release and the levels of NOX4 and p22phox (an essential component of multiple NOX complexes) in wild-type or HSF1-deficient MEFs. The levels of HSF1 and PAI-1 were increased by glyLDL in wild-type MEFs, but not in HSF1-deficient MEFs. Diphenyleneiodonium (a nonspecific NOX inhibitor) or small interfering RNA for p22phox prevented glyLDL-induced increases in the levels of NOX4, HSF1, or PAI-1 in MEFs. The amounts of NOX4, HSF1, and PAI-1 were elevated in hearts of db/db diabetic mice compared to wild-type mice. The results suggest that glyLDL increased the abundance of NOX4 or p22phox via an HSF1-independent pathway, but that of PAI-1 via an HSF1-dependent manner. NOX4 plays a crucial role in glyLDL-induced expression of HSF1 and PAI-1 in mouse fibroblasts. Increased expression of NOX4, HSF1, and PAI-1 was detected in cardiovascular tissue of diabetic mice.     

Deranged aortic intima-media thickness, plasma triglycerides and granulopoiesis in Sl/Sl(d) mice

Studies were carried out to evaluate the impact of a high-fat dietary regimen on aortic wall thickness, peripheral blood leukocyte profile, and plasma cholesterol and triglyceride levels in the mast cell-deficient Sl/Sl(d) mouse. The results demonstrated that the mean aortic wall thickness of Sl/Sl(d) mice was significantly higher than their normal littermates, and were increased in both genotypes after a 17-day high-fat regimen. In comparison with normal littermates, Sl/Sl(d) genotypes had elevated levels of plasma triglycerides with normal levels of plasma cholesterol, and the high-fat diet markedly lowered the triglyceride levels. Total peripheral blood leukocytes, the monocyte and granulocyte counts, and hemoglobin levels were significantly lower in Sl/Sl(d) mice, although the number of lymphocytes, eosinophils and basophils were the same in both genotypes. Interestingly, the high-fat diet regimen elevated leukocyte counts and the number of monocytes and granulocytes in Sl/Sl(d) mice.     

Association analysis of the plasminogen activator inhibitor-1 4G/5G polymorphism in Hispanics and African Americans: the IRAS family study

OBJECTIVE: Plasminogen activator inhibitor type-1 (PAI-1) plays a central role in fibrolysis and has recently been hypothesized to influence components of the insulin resistance syndrome. We consider whether the 4G/5G polymorphism influences components of insulin resistance and obesity solely through PAI-1 protein levels or also though a secondary pathway. In addition, we explore whether transforming growth factor (TGF-beta1), a key regulator of PAI-1 expression, modifies the influence of the PAI-1 4G/5G polymorphism on these traits. METHODS AND RESULTS: The Insulin Resistance and Atherosclerosis (IRAS) Family Study genotyped 287 African American (18 pedigrees) and 811 Hispanic American (45 pedigrees) individuals for the 4G/5G PAI-1 and two TGF-beta1 polymorphisms (R25P, C-509T). Individuals were recruited from three clinical centers located in San Antonio (urban Hispanic), San Luis Valley (rural Hispanic) and Los Angeles (African American). The presence of the 4G PAI-1 allele was positively associated with PAI-1 protein level (combined sample p < 0.0001). Hispanic Americans average 65% higher PAI-1 protein levels than African Americans (p < 0.0001). Consistently across ethnic groups, increased PAI-1 protein levels were associated with increased insulin resistance and overall and central obesity (p value < 0.0001, combined sample). Adjusting for PAI-1 protein levels, there was evidence of an association of PAI-1 genotype (4G) with insulin sensitivity (p < 0.002) and subcutaneous fat (p < 0.01). These associations were not influenced by TGF-beta1 genotypes. CONCLUSIONS: PAI-1 protein is a strong correlate of insulin resistance (IR) and obesity in Hispanics and African Americans. However, PAI-1 4G/5G polymorphism appears to influence insulin resistance and obesity beyond its direct influence on serum PAI-1 protein levels.     

Increased PAI-1 in females compared with males is protective for abdominal aortic aneurysm formation in a rodent model

The serine proteases, along with their inhibitor plasmin activator inhibitor-1 (PAI-1), have been shown to play a role in abdominal aortic aneurysm (AAA) formation. The aim of this study is to determine if PAI-1 may be a protective factor for AAA formation and partially responsible for the gender difference observed in AAAs. Male and female wild-type (WT) C57BL/6 and PAI-1(-/-) mice 8-12 wk of age underwent aortic perfusion with porcine pancreatic elastase. Animals were harvested 14 days following perfusion and analyzed for phenotype, PAI-1 protein levels, and matrix metalloproteinase (MMP)-9 and -2 activity. WT males had an average increase in aortic diameter of 80%, whereas females only increased 32% (P < 0.001). PAI-1(-/-) males increased 204% and females 161%, significantly more than their WT counterparts (P < 0.001). Western blot revealed 61% higher PAI-1 protein levels in the WT females compared with the WT males (P = 0.01). Zymography revealed higher levels of pro-MMP-2 and active MMP-2 in the PAI-1(-/-) males and females compared with their WT counterparts. PAI-1(-/-) females had significantly higher serum plasmin levels compared with WT females (P = 0.003). In conclusion, WT female mice are protected from aneurysm formation and have higher levels of PAI-1 compared with males during experimental aneurysm formation. Additionally, both male and female PAI-1(-/-) animals develop significantly larger aneurysms than WT animals, correlating with higher pro- and active MMP-2 levels. These findings suggest that PAI-1 is protective for aneurysm formation in the elastase model of AAA and plays a role in the gender differences seen in AAA formation.     

Inhibition of apoptosis and caspase-3 in vascular smooth muscle cells by plasminogen activator inhibitor type-1

Increased expression of plasminogen activator inhibitor type 1 (PAI-1) is associated with decreased apoptosis of neoplastic cells. We sought to determine whether PAI-1 alters apoptosis in vascular smooth muscle cells (VSMC) and, if so, by what mechanisms. A twofold increase in the expression of PAI-1 was induced in VSMC from transgenic mice with the use of the SM-22alpha gene promoter (SM22-PAI+). Cultured VSMC from SM22-PAI+ mice were more resistant to apoptosis induced by tumor necrosis factor plus phorbol myristate acetate or palmitic acid compared with VSMC from negative control littermates. Both wild type (WT) and a stable active mutant form of PAI-1 (Active) inhibited caspase-3 amidolytic activity in cell lysates while a serpin-defective mutant (Mut) PAI-1 did not. Similarly, both WT and Active PAI-1 decreased amidolytic activity of purified caspase-3, whereas Mut PAI-1 did not. WT but not Mut PAI-1 decreased the cleavage of poly-[ADP-ribose]-polymerase (PARP), the physiological substrate of caspase-3. Noncovalent physical interaction between caspase-3 and PAI-1 was demonstrable with the use of both qualitative and quantitative in vitro binding assays. High affinity binding was eliminated by mutations that block PAI-1 serpin activity. Accordingly, attenuated apoptosis resulting from elevated expression of PAI-1 by VSMC may be attributable, at least in part, to reversible inhibition of caspase-3 by active PAI-1.     

Increased plasminogen activator inhibitor type-1 (PAI-1) in the heart as a function of age

Heart failure is associated with advanced age and insulin resistance and is thought to be exacerbated by cardiac fibrosis. Plasminogen activator inhibitor type-1 (PAI-1) has been strongly implicated as a determinant of fibrosis in diverse organs and tissues. Its concentration is increased in blood, and its expression is increased in vessel walls in association with insulin resistance. Accordingly, we sought to determine whether expression of PAI-1 in the heart increases as a function of age of 10 week old and 20 week old normal and insulin resistant transgenic mice thereby potentially predisposing to heart failure. Results obtained indicate that PAI-1 content increases significantly in the heart as a function of age by more than 60%. The increases are much greater than those that can be accounted for by the modest, and statistically insignificant increases in the concentrations of PAI-1 in plasma that were observed to occur as a function of age as well. Thus, PAI-1 increases in the heart is a function of age, occurs in insulin resistant and non-insulin resistant mice, and may contribute to fibrosis predisposing to heart failure associated with advanced age, particularly when insulin resistance is present.     

Serp-1, a viral anti-inflammatory serpin,regulates cellular serine proteinase and serpin responses to vascular injury

Complex DNA viruses have tapped into cellular serpin responses that act as key regulatory steps in coagulation and inflammatory cascades. Serp-1 is one such viral serpin that effectively protects virus-infected tissues from host inflammatory responses. When given as purified protein, Serp-1 markedly inhibits vascular monocyte invasion and plaque growth in animal models. We have investigated mechanisms of viral serpin inhibition of vascular inflammatory responses. In vascular injury models, Serp-1 altered early cellular plasminogen activator (tissue plasminogen activator), inhibitor (PAI-1), and receptor (urokinase-type plasminogen activator) expression (p < 0.01). Serp-1, but not a reactive center loop mutant, up-regulated PAI-1 serpin expression in human endothelial cells. Treatment of endothelial cells with antibody to urokinase-type plasminogen activator and vitronectin blocked Serp-1-induced changes. Significantly, Serp-1 blocked intimal hyperplasia (p < 0.0001) after aortic allograft transplant (p < 0.0001) in PAI-1-deficient mice. Serp-1 also blocked plaque growth after aortic isograft transplant and after wire-induced injury (p < 0.05) in PAI-1-deficient mice indicating that increase in PAI-1 expression is not required for Serp-1 to block vasculopathy development. Serp-1 did not inhibit plaque growth in uPAR-deficient mice after aortic allograft transplant. We conclude that the poxviral serpin, Serp-1, attenuates vascular inflammatory responses to injury through a pathway mediated by native uPA receptors and vitronectin.     

The MMP-9/TIMP-1 System is Involved in Fluoride-Induced Reproductive Dysfunctions in Female Mice

A total of 84 healthy female mice were kept with various concentrations of sodium fluoride (F) (0, 50, 100, 150 mg F^?/L in drinking water for 90 days) and were then mated with healthy male mice for 1 week to study the effect of excessive fluoride on female reproductive function, particularly in embryo implantation. The rate of pregnancy, litter size, and the birth weight of female mice were evaluated. Ultrastructural changes of uteri tissues were observed by transmission electron microscopy (TEM). The mRNA expression levels of MMP-9 and TIMP-1 were determined by quantitative real-time PCR. The protein expression levels of MMP-9 and TIMP-1 were analyzed by western blotting. Results showed a significant decrease of litter size in mice exposed to fluoride. TEM images of uteri tissue of mice that underwent a 150 mg/L F^? treatment for 90 days showed a vague nucleus, reduced microvilli, increased lysosomes, a dilated endoplasmic reticulum, and a vacuolization mitochondrion when compared with the control group. Following the damage of the structure, the expression levels of MMP-9 and TIMP-1 in uteri tissues were significantly unregulated in the F 150 group. These results show that MMP-9/TIMP-1 system disturbance and changes of histological structure in uteri tissue are involved in fluoride-induced reproductive dysfunctions.