Publications

OBJECTIVE

Resolution of deep venous thrombosis (DVT) is involved in the pathogenesis of postthrombotic syndrome. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that are critical in angiogenesis and tissue remodeling. We hypothesized that MMP-2 and its membrane-bound activator membrane type-1 matrix metalloproteinase (MT1-MMP) expression would be expressed and activated during the resolution of DVT.

METHODS

DVT was generated by caval ligation in wild-type and MMP-2 transgenic reporter mice. Ligated and sham-operated (control) cavae were analyzed for MMP-2 transcription (beta-galactosidase activity in MMP-2 reporter mice) and MT1-MMP mRNA by real-time polymerase chain reaction. MMP-2 activity was determined by zymography, and immunohistochemical staining for beta-galactosidase and MT1-MMP protein was used to localize expression. Human umbilical vascular endothelial cells (HUVEC) were treated with 10 U/mL thrombin and MMP-2 and MT1-MMP mRNA levels and MMP-2 activity was determined.

RESULTS

MMP-2 activity increased 71% (n = 5, P < .05) at day 8 in ligated vs control cavae by zymography. beta-galactosidase activity showed a 1.2-fold (n = 8, P < .05) and 1.7-fold (n = 8, P < .05) induction in MMP-2 transcription at day 3 and day 8, respectively. No significant MT1-MMP gene induction was seen at day 3 in ligated vs control cavae, but MT1-MMP mRNA was upregulated 2.5-fold (n = 8, P < .05) in ligated cavae at day 8. Immunohistochemical staining localized MMP-2 and MT1-MMP expression to the vein wall and cellular infiltrates of the thrombus. Thrombin-treated HUVEC showed differential responses of MMP-2 and MT1-MMP. Zymography of conditioned media and cell lysates illustrated a 220% (152.6 +/- 8.6 vs 69.445 +/- 5.46 pixels/unit area, n = 5, P < .05) and 150% (74.1 +/- 7.3 vs 49.2 +/- 5.7 pixels/unit area, n = 5, P < .05) increases in MMP-2 activity respectively. MMP-2 mRNA levels were downregulated 30% (0.48 +/- 0.023 vs 0.63 +/- 0.035 copies of MMP-2 mRNA/copy GAPDH, n = 5, P < .05), whereas MT1-MMP message was upregulated 250% (0.147 +/- 0.009 vs 0.059 +/- 0.005 copies of MT1-MMP mRNA/copy GAPDH, n = 5, P < .05).

CONCLUSIONS

Resolution of DVT is associated with increased MMP-2 transcription and activity as well as MT1-MMP expression. Thrombin may mediate the increase in MT1-MMP noted in DVT. This is the first article studying MMP-2 and MT1-MMP transcription in DVT. These findings add DVT resolution to the class of inflammatory and fibrotic disorders in which transcriptional activation of the MT1-MMP/MMP-2 genes occurs and identify a potential therapeutic target to modulate this clinically relevant process.

CLINICAL RELEVANCE

Postthrombotic syndrome remains a significant clinical problem after deep venous thrombosis (DVT), but the cellular and molecular mechanisms involved in thrombus resolution and vein wall fibrosis remain undefined. Matrix metalloproteinase (MMP) enzymes are critical to cell migration and matrix breakdown. We identify gene transcription and activity of two MMP isoforms, MMP-2 and MMP-14 (membrane type MMP 1, MT1-MMP) in the resolution phase of experimental DVT and in thrombin-treated endothelial cells. These studies define new proteases potentially important to resolution of DVT and development of postthrombotic syndrome.

Members of the integrin family recognize a variety of spatially-restricted extracellular ligands. Classically, ligation of integrins activates cytoplasmic signals in the integrin-expressing cell and contributes to cell adhesion, migration, proliferation and survival. At least two members of this family, alphavbeta6 and alphavbeta8 perform an additional function, activation of latent complexes of transforming growth factor beta. In effect, this process allows integrins on one cell to activate signals on adjacent (in the case of alphavbeta6) or nearby cells (in the case of alphavbeta8). Integrin-mediated TGFbeta activation has been shown to play important roles in modulating tissue fibrosis, acute lung injury and pulmonary emphysema. Given the important roles that TGFbeta plays in modulating epithelial cell growth, epithelial-to-mesenchymal transformation and tumor invasion and metastasis, integrin-mediated TGFbeta activation is likely to play important roles in tumor growth and metastasis.

BACKGROUND

Preovulatory gonadotropin-releasing hormone and luteinizing hormone (LH) surges depend on activation of estrogen-inducible progesterone receptors (PRs) in the hypothalamus. Although testosterone treatment can suppress LH secretion under some circumstances, how androgens affect the release of preovulatory hormone surges, and the cellular mechanisms by which androgens exert any such effects, remains unknown.

OBJECTIVE

This study examined the hypothesis that testosterone can block the release of estrogen-induced gonadotropin surges via attenuation of estrogen's ability to induce PRs in the preoptic area (POA)-hypothalamus.

METHODS

In experiment 1, proestrus rats were implanted with capsules filled with crystalline testosterone or empty control capsules. Four days later, animals were bled via atrial catheters at 30-minute intervals from noon to 9:00 pm. In experiment 2, proestrus rats received testosterone-filled or empty control capsules, and 3 days later were ovariectomized (OVX) and injected with estradiol benzoate (EB) 30 mug SC or sesame oil vehicle. The next day, blood samples were obtained from the rats. In experiment 3, proestrus rats similarly implanted with testosterone-filled or empty control capsules, OVX, and injected with EB or vehicle were sacrificed, and POA-hypothalamic tissue was collected for quantitative reverse transcription-polymerase chain reaction analysis of PR messenger RNA.

RESULTS

In experiment 1, radioimmunoassay of serum revealed that testosterone completely blocked release of LH surges that were fully evident in the control group. In experiment 2, LH radioimmunoassay revealed that high-physiologic testosterone exposure completely abolished the release of EB-induced LH surges. In experiment 3, although EB treatment was found to induce an increase in PR expression in control animals, no such induction of PR expression was observed in the testosterone-treated rats.

CONCLUSIONS

Our findings are consistent with the hypothesis that hyperandrogenic interference in the release of preovulatory LH surges is mediated by the suppressive effects of androgens on PR expression in POA-hypothalamic tissue. These findings may have important implications in the understanding of reproductive dysregulation in female hyperandrogenic syndromes, including polycystic ovary syndrome.

The role of NK cells following solid organ transplantation remains unclear. We examined NK cells in acute allograft rejection using a high responder model (DA-->Lewis) of rat orthotopic liver transplantation. Recipient-derived NK cells infiltrated liver allografts early after transplantation. Since chemokines are important in the trafficking of cells to areas of inflammation, we determined the intragraft expression of chemokines known to attract NK cells. CCL3 was significantly increased in allografts at 6 h post-transplant as compared to syngeneic grafts whereas CCL2 and CXCL10 were elevated in both syngeneic and allogeneic grafts. CXCL10 and CX3CL1 were significantly upregulated in allografts by day 3 post-transplant as compared to syngeneic grafts suggesting a role for these chemokines in the recruitment of effector cells to allografts. Graft-infiltrating NK cells were shown to be a major source of IFN-gamma, and IFN-gamma levels in the serum were markedly increased, specifically in allograft recipients, by day 3 post-transplant. Accordingly, in the absence of NK cells the levels of IFN-gamma were significantly decreased. Furthermore, graft survival was significantly prolonged. These data suggest that IFN-gamma-producing NK cells are an important link between the innate and adaptive immune responses early after transplantation.

The role of cytotoxic T-lymphocyte (CTL) escape in rapidly progressive infant human immunodeficiency virus type 1 (HIV-1) infection is undefined. The data presented here demonstrate that infant HIV-1-specific CTL can select for viral escape variants very early in life. These variants, furthermore, may be selected specifically in the infant, despite the same CTL specificity being present in the mother. Additionally, pediatric CTL activity may be compromised both by the transmission of maternal escape variants and by mother-to-child transmission of escape variants that originally arose in the father. The unique acquisition of these CTL escape forms may help to explain the severe nature of some pediatric HIV infections.

Formation and transport of glucuronide metabolites were studied in LLC-PK1 cells. Glucuronidation of 17beta-estradiol, 1-naphthol, mycophenolic acid, and 4-methylumbelliferone was examined in microsomes prepared from LLC-PK1 cells, human livers, human kidneys, and human intestines. The rate of glucuronide metabolite formation observed with LLC-PK1 microsomes was comparable to rates observed with various human tissue microsomes. The fate of the glucuronide metabolite formed in the LLC-PK1 cells was studied by examining its extracellular transport using mycophenolic acid as a model substrate. After administration of mycophenolic acid, the amount of the glucuronide metabolite exiting to the extracellular compartments significantly decreased in the presence of MK-571, an inhibitor for the multidrug resistance-associated protein (MRP) transporter. However, the intracellular levels of the glucuronide metabolite did not change, suggesting that MK-571 was probably blocking metabolite efflux. In summary, these results suggest that the glucuronidating enzyme(s) expressed in the LLC-PK1 cells are capable of sufficient glucuronidation activity and that endogenous transporter(s) in LLC-PK1 cells are active and determine the distribution of the formed metabolites. Since these cells have been previously used to study drug transport, they may be a useful tool in future studies to explore the effect of drug transporters on glucuronidation.

Major depression is associated with adverse outcomes in patients who have coronary heart disease. How best to identify depression in busy cardiology practices is unknown. We compared the test characteristics of 4 depression screening instruments with an interview diagnosis of depression (Diagnostic Interview Schedule) in 1,024 outpatients who had coronary heart disease. Screening instruments were the 10-item Center for Epidemiologic Studies Depression Scale-10, the Patient Health Questionnaire-9, the Patient Health Questionnaire-2, and a simple 2-item instrument that asks (1) "During the past month, have you often been bothered by feeling down, depressed, or hopeless?" and (2) "During the past month, have you often been bothered by little interest or pleasure in doing things?" Of the 1,024 participants, 224 (22%) had major depression based on the Diagnostic Interview Schedule. Areas under the receiver-operating characteristic curves were similar for all instruments (range 0.84 to 0.87). In conclusion, a positive response to 1 of the 2 items was 90% sensitive and 69% specific for depression, with a negative likelihood ratio of 0.14. Thus, negative responses to the 2 items effectively ruled out depression. A score > or =10 on the Patient Health Questionnaire-9 was 54% sensitive and 90% specific, with a positive likelihood ratio of 5.4. Thus, a cutpoint > or =10 was virtually diagnostic for depression.

BACKGROUND

Ventricular assist devices (VADs) are important bridges to cardiac transplantation. VAD support may also function as a bridge to ventricular recovery (BTR); however, clinical predictors of recovery and long-term outcomes remain uncertain. We examined the prevalence, characteristics, and outcomes of BTR subjects in a large single center series.

METHODS AND RESULTS

We implanted VADs in 154 adults at the University of Pittsburgh from 1996 through 2003. Of these implants, 10 were BTR. This included 2/80 (2.5%) ischemic patients (supported 42 and 61 days, respectively). Both subjects had surgical revascularization, required perioperative left VAD support, and were alive and transplant-free at follow up (232 and 1319 days, respectively). A larger percentage of nonischemic patients underwent BTR (8/74, 11%; age 30+/-14; 88% female; left ventricular ejection fraction 18+/-6%; supported 112+/-76 days). Three had myocarditis, 4 had post-partum cardiomyopathy (PPCM), and 1 had idiopathic cardiomyopathy. Five received biventricular support. After explantation, ventricular function declined in 2 PPCM patients who then required transplantation. Ventricular recovery in the 6 nonischemic patients surviving transplant-free was maintained (left ventricular ejection fraction 54+/-5%; follow-up 1.5+/-0.9 years). Overall, 8 of 10 BTR patients are alive and free of transplant (follow-up 1.6+/-1.1 years).

CONCLUSIONS

In a large single center series, BTR was evident in 11% of nonischemic patients, and the need for biventricular support did not preclude recovery. For most BTR subjects presenting with acute inflammatory cardiomyopathy, ventricular recovery was maintained long-term. VAD support as BTR should be considered in the care of acute myocarditis and PPCM.