Large-Area Epitaxial Mott Insulating 1T-TaSe2 Monolayer on GaP(111)B.

Two-dimensional Mott materials have recently been reported in the dichalcogenide family with high potential for Mottronic applications. Nevertheless, their widespread use as a single or few layers is hampered by their limited device integration resulting from their growth on graphene, a metallic substrate. Here, we report on the fabrication of 1T-TaSe2 monolayers grown by molecular beam epitaxy on semiconducting gallium phosphide substrates. At the nanoscale, the charge density wave reconstruction and a moiré pattern resulting from the monolayer interaction with the substrate are observed by scanning tunneling microscopy. The fully open gap unveiled by tunneling spectroscopy, which can be further manipulated by the proximity of a metal tip, is confirmed by transport measurements from micrometric to millimetric scales, demonstrating a robust Mott insulating phase at up to 400 K.

Outcomes of renal replacement therapy in the critically ill with COVID-19.

OBJECTIVE: To describe outcomes of critically ill patients with COVID-19, particularly the association of renal replacement therapy to mortality. DESIGN: A single-center prospective observational study was carried out. SETTING: ICU of a tertiary care center. PATIENTS: Consecutive adults with COVID-19 admitted to the ICU. INTERVENTION: Renal replacement therapy. MAIN VARIABLES OF INTEREST: Demographic data, medical history, illness severity, type of oxygen therapy, laboratory data and use of renal replacement therapy to generate a logistic regression model describing independent risk factors for mortality. RESULTS: Of the total of 166 patients, 51% were mechanically ventilated and 26% required renal replacement therapy. The overall hospital mortality rate was 36%, versus 56% for those requiring renal replacement therapy, and 68% for those with both mechanical ventilation and renal replacement therapy. The logistic regression model identified four independent risk factors for mortality: age (adjusted OR 2.8 [95% CI 1.8-4.4] for every 10-year increase), mechanical ventilation (4.2 [1.7-10.6]), need for continuous venovenous hemofiltration (2.3 [1.3-4.0]) and C-reactive protein (1.1 [1.0-1.2] for every 10 mg/L increase). CONCLUSIONS: In our cohort, acute kidney injury requiring renal replacement therapy was associated to a high mortality rate similar to that associated to the need for mechanical ventilation, while multiorgan failure necessitating both techniques implied an extremely high mortality risk.

OBJETIVO: Describir los resultados de pacientes críticamente enfermos con COVID-19, especialmente la asociación de la terapia de reemplazo renal con la mortalidad. DISEÑO: Estudio observacional, prospectivo y unicéntrico. ÁMBITO: En la unidad de cuidados intensivos (UCI) de un centro de atención terciaria. PACIENTES: Pacientes adultos con COVID-19 ingresados de forma consecutiva en la UCI. INTERVENCIÓN: Administración de terapia de reemplazo renal. VARIABLES DE INTERÉS PRINCIPALES: Datos demográficos, antecedentes médicos, gravedad de la enfermedad, tipo de oxigenoterapia, datos analíticos y uso de terapia de reemplazo renal para generar un modelo de regresión logística que describa factores de riesgo independientes de la mortalidad. RESULTADOS: De los 166 pacientes, el 51% recibieron ventilación mecánica (VM) y el 26% requirió terapia de reemplazo renal (TRR). La mortalidad hospitalaria global fue del 36%, frente al 56% en el caso de los pacientes que requirieron TRR y el 68% en el subconjunto de pacientes que necesitó tanto VM como RTT. Un modelo de regresión logística señala cuatro factores de riesgo independientes de la mortalidad: edad (OR ajustada: 2,8 [IC del 95%: 1,8-4,4] por cada incremento de 10 años), ventilación mecánica (4,2 [1,7-10,6]), necesidad de hemofiltración venovenosa continua (HVVC) (2,3 [1,3-4,0]), y proteína C reactiva (1,1 [1,0-1,2] por cada incremento de 10 mg/L). CONCLUSIONES: En nuestra cohorte, la lesión renal aguda que necesita TRR se asocia con una mortalidad similarmente elevada a la de los pacientes que requieren VM, y la insuficiencia multiorgánica que hace necesarias ambas intervenciones se asocia con un riesgo de mortalidad extremadamente alta.

Outcomes of renal replacement therapy in the critically ill with COVID-19 / Resultados de la terapia de reemplazo renal en pacientes críticamente enfermos con COVID-19

Objective To describe outcomes of critically ill patients with COVID-19, particularly the association of renal replacement therapy to mortality. Design A single-center prospective observational study was carried out. Setting ICU of a tertiary care center. Patients Consecutive adults with COVID-19 admitted to the ICU. Intervention Renal replacement therapy. Main variables of interest Demographic data, medical history, illness severity, type of oxygen therapy, laboratory data and use of renal replacement therapy to generate a logistic regression model describing independent risk factors for mortality. Results Of the total of 166 patients, 51% were mechanically ventilated and 26% required renal replacement therapy. The overall hospital mortality rate was 36%, versus 56% for those requiring renal replacement therapy, and 68% for those with both mechanical ventilation and renal replacement therapy. The logistic regression model identified four independent risk factors for mortality: age (adjusted OR 2.8 [95% CI 1.8–4.4] for every 10-year increase), mechanical ventilation (4.2 [1.7–10.6]), need for continuous venovenous hemofiltration (2.3 [1.3–4.0]) and C-reactive protein (1.1 [1.0–1.2] for every 10mg/L increase). Conclusions In our cohort, acute kidney injury requiring renal replacement therapy was associated to a high mortality rate similar to that associated to the need for mechanical ventilation, while multiorgan failure necessitating both techniques implied an extremely high mortality risk. (AU)

Objetivo Describir los resultados de pacientes críticamente enfermos con COVID-19, especialmente la asociación de la terapia de reemplazo renal con la mortalidad. Diseño Estudio observacional, prospectivo y unicéntrico. Ámbito En la unidad de cuidados intensivos (UCI) de un centro de atención terciaria. Pacientes Pacientes adultos con COVID-19 ingresados de forma consecutiva en la UCI. Intervención Administración de terapia de reemplazo renal. Variables de interés principales Datos demográficos, antecedentes médicos, gravedad de la enfermedad, tipo de oxigenoterapia, datos analíticos y uso de terapia de reemplazo renal para generar un modelo de regresión logística que describa factores de riesgo independientes de la mortalidad. Resultados De los 166 pacientes, el 51% recibieron ventilación mecánica (VM) y el 26% requirió terapia de reemplazo renal (TRR). La mortalidad hospitalaria global fue del 36%, frente al 56% en el caso de los pacientes que requirieron TRR y el 68% en el subconjunto de pacientes que necesitó tanto VM como RTT. Un modelo de regresión logística señala cuatro factores de riesgo independientes de la mortalidad: edad (OR ajustada: 2,8 [IC del 95%: 1,8-4,4] por cada incremento de 10 años), ventilación mecánica (4,2 [1,7-10,6]), necesidad de hemofiltración venovenosa continua (HVVC) (2,3 [1,3-4,0]), y proteína C reactiva (1,1 [1,0-1,2] por cada incremento de 10mg/L). Conclusiones En nuestra cohorte, la lesión renal aguda que necesita TRR se asocia con una mortalidad similarmente elevada a la de los pacientes que requieren VM, y la insuficiencia multiorgánica que hace necesarias ambas intervenciones se asocia con un riesgo de mortalidad extremadamente alta. (Au)

Outcomes of renal replacement therapy in the critically ill with COVID-19.

OBJECTIVE: To describe outcomes of critically ill patients with COVID-19, particularly the association of renal replacement therapy to mortality. DESIGN: A single-center prospective observational study was carried out. SETTING: ICU of a tertiary care center. PATIENTS: Consecutive adults with COVID-19 admitted to the ICU. INTERVENTION: Renal replacement therapy. MAIN VARIABLES OF INTEREST: Demographic data, medical history, illness severity, type of oxygen therapy, laboratory data and use of renal replacement therapy to generate a logistic regression model describing independent risk factors for mortality. RESULTS: Of the total of 166 patients, 51% were mechanically ventilated and 26% required renal replacement therapy. The overall hospital mortality rate was 36%, versus 56% for those requiring renal replacement therapy, and 68% for those with both mechanical ventilation and renal replacement therapy. The logistic regression model identified four independent risk factors for mortality: age (adjusted OR 2.8 [95% CI 1.8-4.4] for every 10-year increase), mechanical ventilation (4.2 [1.7-10.6]), need for continuous venovenous hemofiltration (2.3 [1.3-4.0]) and C-reactive protein (1.1 [1.0-1.2] for every 10mg/L increase). CONCLUSIONS: In our cohort, acute kidney injury requiring renal replacement therapy was associated to a high mortality rate similar to that associated to the need for mechanical ventilation, while multiorgan failure necessitating both techniques implied an extremely high mortality risk.

Glial cell line-derived neurotrophic factor (GDNF) induced migration of spermatogonial cells in vitro via MEK and NF-kB pathways.

Glial cell line-derived neurotrophic factor (GDNF) regulates spermatogonial stem cell (SSC) maintenance. In the present study, we examined the levels and the cellular origin of GDNF in mouse testes during age-development, and the capacity of GDNF to induce migration of enriched GFR-α1 positive cells in vitro. The involvement of MAP kinase (MEK) and NF-kB signal pathways were examined. Our results show high levels of GDNF in testicular tissue of one-week-old mice which significantly decreased with age when examined by ELISA, real time PCR (qPCR) and immunofluorescence staining (IF) analysis. GDNF receptor (GFR-α1) expression was similar to GDNF when examined by qPCR analysis. Only Sertoli cell cultures (SCs) from one-week-old mice produced GDNF compared to SCs from older mice. However, peritubular cells from all the examined ages did not produce GDNF. The addition of recombinant GDNF (rGDNF) or supernatant from SCs from one-week-old mice to GFR-α1 positive cells induced their migration in vitro. This effect was significantly reduced by the addition of inhibitors to MEK (PD98059, U0126), NF-kB (PDTC) and IkB protease inhibitor (TPCK). Our results show for the first time the capacity of rGDNF and supernatant from SCs to induce migration of enriched GFR-α1 positive cells, and the possible involvement of MEK, NF-kB and IkB in this process. This study may suggest a novel role for GDNF in the regulation SSC niches and spermatogenesis.

Route of administration-dependent anti-inflammatory effect of liposomal alendronate.

Innate immunity and inflammation are of major importance in various pathological conditions. Intravenous (IV) and intraperitoneal (IP) liposomal alendronate (LA) treatments have been shown to deplete circulating monocytes and peritoneal macrophages resulting in the inhibition of restenosis and endometriosis (EM), respectively. Nevertheless, the correlation between the extent of circulating monocyte depletion and liposome biodistribution is unknown, and the route of administration-dependent bioactivity in restenosis and EM has not been determined. We found that, LA treatment resulted in a dose-response modified biodistribution following both IV and IP administrations. The biodistribution of high-dose LA (10mg/kg), but not that of the low-dose (1mg/kg), was similar in healthy and diseased animals. It is concluded that LA impedes its own elimination from the circulation by depleting circulating monocytes and/or inhibiting their endocytic activity, in a dose-dependent manner. Both IV and IP administration of LA mediated by the partial and transient depletion of circulating monocytes effected inhibition of restenosis. Inhibition of EM was effected only by IP administration, which depleted both intraperitoneal and circulating monocytes. Thus, EM should be considered as a local inflammatory condition with systemic manifestations as opposed to restenosis, a systemic inflammatory disease.

Peritoneal macrophage depletion by liposomal bisphosphonate attenuates endometriosis in the rat model.

BACKGROUND: Activation of macrophages is central to the implantation of endometriosis (EM). We examined the hypothesis that macrophage depletion by intraperitoneal (IP) injection of liposomal alendronate (LA) could result in EM attenuation in a rat model, thus supporting the notion of the pivotal role of macrophages in EM pathology. METHODS: In this study, 90 rats were subjected to an EM model and were divided randomly into seven groups: five groups were treated by 4x once-weekly IP injections of LA (0.02, 0.1, 1, 5 or 10 mg/kg) and the other two groups received saline injections (control) or empty liposomes. Sham-operated rats also received empty liposomes. Depletion of circulating monocytes was determined by flow cytometry analyzes of blood specimens. Four weeks after the initial surgery, the number, size and weight of implants were recorded, adhesions were graded, macrophage infiltration was assessed and the peritoneal fluid was analyzed for monocyte chemotactic protein 1 (MCP-1) and tumor necrosis factor alpha (TNFalpha). RESULTS: Monocyte depletion following IP LA administration resulted in an inhibitory effect on the initiation and growth of EM implants, as expressed by implantation rate, adhesion scoring, implants' size and weight (>0.1 mg/kg LA, P < 0.05). Reduced numbers of infiltrating macrophages were observed in implants of the 1 mg/kg LA group. Peritoneal fluid MCP-1 levels were negatively correlated with LA dose (P < 0.001), whereas no significant correlation could be found for TNFalpha. CONCLUSIONS: Macrophage depletion using IP LA has been shown to effectively inhibit the initiation and growth of EM implants, in a rat EM model. The clear dose-response effect may be viewed as a confirmation of the validity of the concept and encourages further study.

Diabetes associated cell stress and dysfunction: role of mitochondrial and non-mitochondrial ROS production and activity.

It is now widely accepted, given the current weight of experimental evidence, that reactive oxygen species (ROS) contribute to cell and tissue dysfunction and damage caused by glucolipotoxicity in diabetes. The source of ROS in the insulin secreting pancreatic beta-cells and in the cells which are targets for insulin action has been considered to be the mitochondrial electron transport chain. While this source is undoubtably important, we provide additional information and evidence for NADPH oxidase-dependent generation of ROS both in pancreatic beta-cells and in insulin sensitive cells. While mitochondrial ROS generation may be important for regulation of mitochondrial uncoupling protein (UCP) activity and thus disruption of cellular energy metabolism, the NADPH oxidase associated ROS may alter parameters of signal transduction, insulin secretion, insulin action and cell proliferation or cell death. Thus NADPH oxidase may be a useful target for intervention strategies based on reversing the negative impact of glucolipotoxicity in diabetes.

Pleiotropic effects of fatty acids on pancreatic beta-cells.

Hyperlipidemia is frequently associated with insulin resistance states as found in type 2 diabetes and obesity. Effects of free fatty acids (FFA) on pancreatic beta-cells have long been recognized. Acute exposure of the pancreatic beta-cell to FFA results in an increase of insulin release, whereas a chronic exposure results in desensitization and suppression of secretion. We recently showed that palmitate augments insulin release in the presence of non-stimulatory concentrations of glucose. Reduction of plasma FFA levels in fasted rats or humans severely impairs glucose-induced insulin release. These results imply that physiological plasma levels of FFA are important for beta-cell function. Although, it has been accepted that fatty acid oxidation is necessary for its stimulation of insulin secretion, the possible mechanisms by which fatty acids (FA) affect insulin secretion are discussed in this review. Long-chain acyl-CoA (LC-CoA) controls several aspects of the beta-cell function including activation of certain types of protein kinase C (PKC), modulation of ion channels, protein acylation, ceramide- and/or nitric oxide (NO)-mediated apoptosis, and binding to nuclear transcriptional factors. The present review also describes the possible effects of FA on insulin signaling. We showed for the first time that acute exposure of islets to palmitate upregulates the intracellular insulin-signaling pathway in pancreatic islets. Another aspect considered in this review is the source of FA for pancreatic islets. In addition to be exported to the medium, lipids can be transferred from leukocytes (macrophages) to pancreatic islets in co-culture. This process consists an additional source of FA that may plays a significant role to regulate insulin secretion.

Automated sample preparation and gas chromatographic-mass spectrometric analysis of urinary androgenic anabolic steroids.

This paper presents an automated method for extracting anabolic agents from urine samples for their GC-MS analysis by selected-ion monitoring. The sample preparation was carried out in a Hewlett-Packard 7686 SPE PrepStation system. Each 0.6-ml aliquot was hydrolyzed, extracted, dried and trimethylsilyl (TMS) derivatized in a 2-ml vial without any hands-on labor. When sample preparation was finished 2 microl of the extract was injected into the gas chromatograph by split (1:10) mode. Due to the small amount of free space in the 2-ml vials for handling the sample, parameters like time of hydrolysis, type of shaking, number of extractions and some TMS derivatization parameters had to be adjusted to achieve the best recovery for all of the compounds in the screening. Manual and automated sample preparation schemes were compared in terms of linearity, precision, accuracy, limit of detection and recovery data. When large concentrations were analyzed using the automated method no carry-over effect was observed.

Characterization of the mUBC9-binding sites required for E2A protein degradation.

Mammalian Ubc9 (mUbc9) is required for rapid degradation of the E2A proteins E12 and E47 by the ubiquitin-proteasome system. We have shown elsewhere that mUbc9 interacts with amino acids 477-530 of E12/E47. Here we test the hypothesis that this region, rich in proline, glutamic acid, serine, and threonine (PEST) residues, serves as the E2A protein degradation domain (DD). An E2A protein lacking this region, E47Delta(478-531), was significantly more stable than wild-type E47(half-life of more than 6 h versus 55 min). Deletion of the E2A DD had no effect on the E-box-binding and transcriptional activity of E47. We mapped two discreet mUbc9-interacting regions within the E2A DD: amino acids 476-494 and 505-513. E2A(505-513) interacted with mUbc9 but not with human Ubc5, MyoD, Id3, or the polymyositis-scleroderma autoantigen. Substitution of the E2A(505-513) central hydrophobic residues with basic residues abolished interaction with mUbc9. Also, full-length E47 lacking the second mUbc9-interacting region was significantly more stable than wild-type E47. Reintroduction of the E2A DD into the long-lived, naturally occurring chimeric oncoprotein E2A-HLF (hepatic leukemic factor) destabilized it, suggesting that this domain can transfer a degradation signal to a heterologous protein. E2A-HLF-DD chimeric protein was stabilized by the proteasome inhibitor LLNL, indicating the role of the ubiquitin-proteasome system mediating degradation through the E2A degradation domain. Our experiments indicate that the E2A DD mediates E2A protein interactions with the ubiquitin-proteasome system and that the E2A DD is required for metabolism of these widely expressed proteins.

Plasminogen is not required for neointima formation in a mouse model of vein graft stenosis.

Recent studies of mice that lack plasminogen have identified a critical role for this zymogen in arterial remodeling. To permit the use of these (and other) genetically modified mice in the analysis of venous injury, we developed a model in which a patch cut from the external jugular vein of a mouse is grafted to repair a surgically created defect in its carotid artery. In wild-type mice, the venous graft showed initial endothelial denudation and formation of a neointima that progressively and reproducibly expanded in a manner analogous to human vein graft disease, albeit at an accelerated pace. This neointima occupied 37+/-4.6% of the vessel lumen at day 7 and 66+/-5.7% at day 20. The proliferative index of neointimal cells assessed by proliferating cell nuclear antigen staining was 50.6+/-3. 6% at day 7 and 15.2+/-2.0% at day 20. CD45-positive leukocytes and alpha-actin-positive smooth muscle cells accounted for 9.5+/-1.0% and 9.9+/-1.1% of intimal area at day 7, respectively, with the latter increasing to 40.9+/-2.6% at day 20. Collagen accounted for 6.8+/-0.7% of intimal area at day 7 and 20.7+/-1.8% at day 20. Surprisingly, even though arterial neointima formation due to electrostatic and immune-mediated injury is impaired in plasminogen -/- mice, in our study vein graft neointima formation in these mice was not significantly different from that in controls (70.9+/-6.4 versus 65.6+/-4.4% luminal occlusion, P=NS). Thus, plasmin proteolysis, although critical in extracellular matrix degradation and cellular migration after arterial injury, does not appear to be so important in vein graft neointima formation, perhaps because of the relative lack of structural barriers to cellular migration in the normal vein wall. This novel model of vein graft injury should be useful for further studies of differences in the response to injury of arterial and venous tissues.

Interferon-gamma-mediated inhibition of cyclin A gene transcription is independent of individual cis-acting elements in the cyclin A promoter.

Interferons (IFNs) affect cellular functions by altering gene expression. The eukaryotic cell cycle is governed in part by the periodic transcription of cyclin genes, whose protein products associate with and positively regulate the cyclin-dependent kinases. To understand better the growth inhibitory effect of IFN-gamma on vascular smooth muscle cells (VSMCs), we compared the expression and activity of G1 and S phase cyclins in control and IFN-gamma-treated VSMCs. IFN-gamma treatment did not inhibit the G1 cyclins but did decrease cyclin A protein, mRNA, and associated kinase activity by 85, 90, and 90%, respectively. Nuclear run-on and mRNA stability determinations indicated that this decrease was the result of transcriptional inhibition. To investigate the molecular basis of this inhibition, we examined protein-DNA interactions involving the cyclin A promoter. Electromobility shift assays showed little change with IFN-gamma treatment in the binding of nuclear proteins to isolated ATF, NF-Y, and CDE elements. In vivo genomic footprinting indicated that IFN-gamma treatment changed the occupancy of chromosomal NF-Y and CDE sites slightly and did not affect occupancy of the ATF site. In a previous study of transforming growth factor-beta1-mediated inhibition of the cyclin A promoter, we mapped the inhibitory effect to the ATF site; in the present study of IFN-gamma treatment, functional analysis by transient transfection showed that inhibition of the cyclin A promoter persisted despite mutation of the ATF, NF-Y, or CDE elements. We hypothesize that IFN-gamma inhibits cyclin A transcription by modifying co-activators or general transcription factors within the complex that drives transcription of the cyclin A gene.

Donor MHC and adhesion molecules in transplant arteriosclerosis.

Transplant-associated arteriosclerosis remains an obstacle to long-term graft survival. To determine the contribution to transplant arteriosclerosis of MHC and adhesion molecules from cells of the donor vasculature, we allografted carotid artery loops from six mutant mouse strains into immunocompetent CBA/CaJ recipients. The donor mice were deficient in either MHC I molecules or MHC II molecules, both MHC I and MHC II molecules, the adhesion molecule P-selectin, intercellular adhesion molecule (ICAM)-1, or both P-selectin and ICAM-1. Donor arteries in which ICAM-1, MHC II, or both MHC I and MHC II were absent showed reductions in neointima formation of 52%, 33%, and 38%, respectively, due primarily to a reduction in smooth muscle cell (SMC) accumulation. In P-selectin-deficient donor arteries, neointima formation did not differ from that in controls. In donor arteries lacking both P-selectin and ICAM-1, the size of the neointima was similar to that in those lacking ICAM-1 alone. In contrast, neointima formation increased by 52% in MHC I-deficient donor arteries. The number of CD4-positive T cells increased by 2.8-fold in MHC I-deficient arteries, and that of alpha-actin-positive SMCs by twofold. These observations indicate that ICAM-1 and MHC II molecules expressed in the donor vessel wall may promote transplant-associated arteriosclerosis. MHC I molecules expressed in the donor may have a protective effect.

Use of controlled vignettes in evaluation: does type of response method make a difference?

Two studies were performed using vignettes as a method in program evaluation. One study used a rating scale method. The second study used a forced choice decision method. The two studies were performed with students from the pre-Occupational Therapy (pre-OT) program, the Occupational Therapy (professional OT) program and the College of Business at Colorado State University. Students were asked to respond to a questionnaire about how a hypothetical job applicant with a cognitive disability would perform in one of three types of job settings: a segregated, sheltered workshop setting; an integrated supported employment setting; or an integrated competitive employment setting. The students in the Professional Occupational Therapy program had been part of a training grant, 'An integrated approach to personnel preparation for transition', which focused on the integration of transition competencies into an occupational therapy curriculum. The forced choice vignette method resulted in statistically significant positive outcomes toward supported employment and the influence of the training grant. The discussion focused on the different outcomes of the two studies relative to the methods applied and the use of vignettes as an evaluation tool to assess the impact of a modified curriculum. Attention was directed toward the problem of external validity when using vignettes (Gorman, C.D., Clover, W.H. & Doherty, M.E., (l978). Can we learn anything about interviewing real people from 'interviews' of paper people? Two studies of the external validity of a paradigm. Organizational Behavior and Human Performance, 22, 165-192.).

Reduced transplant arteriosclerosis in plasminogen-deficient mice.

Recent gene targeting studies indicate that the plasminogen system is implicated in cell migration and matrix degradation during arterial neointima formation and atherosclerotic aneurysm formation. This study examined whether plasmin proteolysis is involved in accelerated posttransplant arteriosclerosis (graft arterial disease). Donor carotid arteries from wild-type B10.A2R mice were transplanted into either plasminogen wild-type (Plg+/+) or homozygous plasminogen-deficient (Plg-/-) recipient mice with a genetic background of 75% C57BL/6 and 25% 129. Within 15 d after allograft transplantation, leukocytes and macrophages infiltrated the graft intima in Plg+/+ and Plg-/- recipient mice to a similar extent. In Plg+/+ recipients, the elastic laminae in the transplant media exhibited breaks through which macrophages infiltrated before smooth muscle cell proliferation, whereas in Plg-/- recipients, macrophages failed to infiltrate the transplant media which remained structurally more intact. After 45 d of transplantation, a multilayered smooth muscle cell-rich transplant neointima developed in Plg+/+ hosts, in contrast to Plg-/- recipients, in which the transplants contained a smaller intima, predominantly consisting of leukocytes, macrophages, and thrombus. Media necrosis, fragmentation of the elastic laminae, and adventitial remodeling were more pronounced in Plg+/+ than in Plg-/- recipient mice. Expression of the plasminogen activators (PA), urokinase-type PA (u-PA) and tissue-type PA (t-PA), and expression of the matrix metalloproteinases (MMPs), MMP-3, MMP-9, MMP-12, and MMP-13, were significantly increased within 15 d of transplantation when cells actively migrate. These data indicate that plasmin proteolysis plays a major role in allograft arteriosclerosis by mediating elastin degradation, macrophage infiltration, media remodeling, medial smooth muscle cell migration, and formation of a neointima.

Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system.

Proteins of the LIM family are critical regulators of development and differentiation in various cell types. We have described the cloning of cysteine-rich protein 2/smooth muscle LIM protein (CRP2/SmLIM), a LIM-only protein expressed in differentiated vascular smooth muscle cells. As a first step toward understanding the potential functions of CRP2/SmLIM, we analyzed its expression after gastrulation in developing mice and compared the expression of CRP2/SmLIM with that of the other 2 members of the CRP subclass, CRP1 and CRP3/MLP. In situ hybridization in whole-mount and sectioned embryos showed that CRP2/SmLIM was expressed in the sinus venosus and the 2 cardiac chambers at embryonic day 9. Vascular expression of CRP2/SmLIM was first seen at embryonic day 10. At subsequent time points, CRP2/SmLIM expression decreased in the heart but remained high in the vasculature. CRP1 was expressed both in vascular and nonvascular tissues containing smooth muscle cells, whereas CRP3/MLP was expressed only in tissues containing striated muscle. These patterns of expression were maintained in the adult animal and suggest an important role for this gene family in the development of smooth and striated muscle.

Regulation of CD44 gene expression by the proinflammatory cytokine interleukin-1beta in vascular smooth muscle cells.

The CD44 gene codes for a family of alternatively spliced, multifunctional adhesion molecules that participate in extracellular matrix binding, lymphocyte activation, cell migration, and tumor metastasis. In a mouse model of transplant-associated arteriosclerosis, CD44 protein was induced in the neointima of allografted vessels and colocalized with a subset of proliferating vascular smooth muscle cells (SMC). To elucidate the molecular mechanisms regulating CD44 expression in this model, we investigated the regulation of CD44 gene expression by interleukin (IL)-1beta. Treatment of rat aortic SMC with IL-1beta resulted in a 5.3-fold increase in cell surface CD44 expression. Northern analysis showed that IL-1beta promoted a dose- and time-dependent induction of CD44 mRNA which reached 6.6-fold after 48 h, and nuclear run-on analysis showed that IL-1beta increased the rate of CD44 gene transcription within 8 h of stimulation. In transient reporter gene transfection experiments in rat aortic SMC, a 1.4-kilobase fragment of the mouse CD44 5'-flanking sequence mediated this response to IL-1beta. Regulation of CD44 gene expression by the proinflammatory cytokine IL-1beta may contribute to SMC phenotypic modulation in the pathogenesis of arteriosclerosis.

Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation.

Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The approximately 4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.