A cross-institutional analysis of the effects of broadening trainee professional development on research productivity.

PhD-trained scientists are essential contributors to the workforce in diverse employment sectors that include academia, industry, government, and nonprofit organizations. Hence, best practices for training the future biomedical workforce are of national concern. Complementing coursework and laboratory research training, many institutions now offer professional training that enables career exploration and develops a broad set of skills critical to various career paths. The National Institutes of Health (NIH) funded academic institutions to design innovative programming to enable this professional development through a mechanism known as Broadening Experiences in Scientific Training (BEST). Programming at the NIH BEST awardee institutions included career panels, skill-building workshops, job search workshops, site visits, and internships. Because doctoral training is lengthy and requires focused attention on dissertation research, an initial concern was that students participating in additional complementary training activities might exhibit an increased time to degree or diminished research productivity. Metrics were analyzed from 10 NIH BEST awardee institutions to address this concern, using time to degree and publication records as measures of efficiency and productivity. Comparing doctoral students who participated to those who did not, results revealed that across these diverse academic institutions, there were no differences in time to degree or manuscript output. Our findings support the policy that doctoral students should participate in career and professional development opportunities that are intended to prepare them for a variety of diverse and important careers in the workforce.

Trafficking of endogenous smooth muscle cell cholesterol: a role for serum amyloid A and interleukin-1ß.

OBJECTIVE: Intracellular cholesterol distribution impacts cell function; however, processes influencing endogenous cholesterol trafficking remain largely unknown. Atherosclerosis is associated with vascular inflammation and these studies address the role of inflammatory mediators on smooth muscle cell cholesterol trafficking. METHODS AND RESULTS: Interestingly, in the absence of an exogenous cholesterol source, serum amyloid A increased [(14)C] oleic acid incorporation into cholesteryl ester in rat smooth muscle cells, suggesting endogenous cholesterol trafficking to the endoplasmic reticulum. [(3)H] cholesteryl ester accumulated in cells prelabeled with [(3)H] cholesterol, confirming that serum amyloid A mediated the movement of endogenous cholesterol. Cholesterol movement was dependent upon functional endolysosomes. The cholesterol oxidase-sensitive pool of cholesterol decreased in serum amyloid A-treated cells. Furthermore, the mechanism whereby serum amyloid A induced cholesterol trafficking was determined to be via activation of expression of secretory phospholipase A(2), group IIA (sPLA(2)) and sPLA(2)-dependent activation of sphingomyelinase. Interestingly, although neither tumor necrosis factor-α nor interferon-γ induced cholesterol trafficking, interleukin-1ß induced [(14)C] cholesteryl ester accumulation that was also dependent upon sPLA(2) and sphingomyelinase activities. Serum amyloid A activates smooth muscle cell interleukin-1ß expression, and although the interleukin-1-receptor antagonist inhibited the interleukin-1ß-induced cholesterol trafficking, it had no effect on the movement of cholesterol mediated by serum amyloid A. CONCLUSIONS: These data support a role for inflammation in endogenous smooth muscle cell cholesterol trafficking from the plasma membrane to the endoplasmic reticulum.

Secretory phospholipase A2, group IIA is a novel serum amyloid A target gene: activation of smooth muscle cell expression by an interleukin-1 receptor-independent mechanism.

Atherosclerosis is a multifactorial vascular disease characterized by formation of inflammatory lesions. Elevated circulating acute phase proteins indicate disease risk. Serum amyloid A (SAA) is one such marker but its function remains unclear. To determine the role of SAA on aortic smooth muscle cell gene expression, a preliminary screen of a number of genes was performed and a strong up-regulation of expression of secretory phospholipase A(2), group IIA (sPLA(2)) was identified. The SAA-induced increase in sPLA(2) was validated by real time PCR, Western blot analysis, and enzyme activity assays. Demonstrating that SAA increased expression of sPLA(2) heteronuclear RNA and that inhibiting transcription eliminated the effect of SAA on sPLA(2) mRNA suggested that the increase was transcriptional. Transient transfections and electrophoretic mobility shift assays identified CAAT enhancer-binding protein (C/EBP) and nuclear factor kappaB (NFkappaB) as key regulatory sites mediating the induction of sPLA(2). Moreover, SAA activated the inhibitor of NF-kappaB kinase (IKK) in cultured smooth muscle cells. Previous reports showed that interleukin (IL)-1beta up-regulates Pla2g2a gene transcription via C/EBPbeta and NFkappaB. Interestingly, SAA activated smooth muscle cell IL-1beta mRNA expression, however, blocking IL-1 receptors had no effect on SAA-mediated activation of sPLA(2) expression. Thus, the observed changes in sPLA(2) expression were not secondary to SAA-induced IL-1 receptor activation. The association of SAA with high density lipoprotein abrogated the SAA-induced increase in sPLA(2) expression. These data suggest that during atherogenesis, SAA can amplify the involvement of smooth muscle cells in vascular inflammation and that this can lead to deposition of sPLA(2) and subsequent local changes in lipid homeostasis.

The A2b adenosine receptor protects against vascular injury.

The A2b adenosine receptor (A2bAR) is highly abundant in bone marrow macrophages and vascular smooth muscle cells (VSMC). To examine the functional significance of this receptor expression, we applied a femoral artery injury model to A2bAR knockout (KO) mice and showed that the A2bAR prevents vascular lesion formation in an injury model that resembles human restenosis after angioplasty. While considering related mechanisms, we noted higher levels of TNF-alpha, an up-regulator of CXCR4, and of VSMC proliferation in the injured KO mice. In accordance, CXCR4, which is known to attract progenitor cells during tissue regeneration, is up-regulated in lesions of the KO mice. In addition, aortic smooth muscle cells derived from A2bAR KO mice display greater proliferation in comparison with controls. Bone marrow transplantation experiments indicated that the majority of the signal for lesion formation in the null mice originates from bone marrow cells. Thus, this study highlights the significance of the A2bAR in regulating CXCR4 expression in vivo and in protecting against vascular lesion formation.

The A2B adenosine receptor protects against inflammation and excessive vascular adhesion.

Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor-knockout/reporter gene-knock-in (A2BAR-knockout/reporter gene-knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-alpha, and a consequent downregulation of IkappaB-alpha are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.

Improving Diversity of Dental Students Through the Boston University Master's of Oral Health Sciences Postbaccalaureate Program.

Boston University Goldman School of Dental Medicine (GSDM), in collaboration with Boston University School of Medicine, introduced the Oral Health Sciences (OHS) pipeline program in 2005 to enhance the academic preparedness of students from underrepresented minority (URM) groups for dental school admission. The aim of this study was to evaluate the OHS program's success in preparing URM students for dental school, as measured by acceptance to dental school and performance in the first and second years. Data on 2005-15 program enrollees were collected from admissions records, the registrar, and the Office of Institutional Research on students' race/ethnicity, undergraduate and OHS grade point average (GPA), and Dental Admission Test (DAT) scores. Acceptance to dental school and performance at GSDM for non-URM OHS graduates, URM OHS graduates, and non-OHS dental students were compared. A total of 55 URM students completed the OHS program during this period, with 49 successfully matriculating to a dental school in the U.S. and 33 attending GSDM. Average OHS GPA was higher for those URM students accepted to dental school than for those who did not gain admission (3.36±0.30 vs. 2.94±0.19). Evaluation of the academic performance of URM OHS students in the first year (p=0.13) and second year (p=0.88) at GSDM showed that these students performed as well as the non-OHS and non-URM OHS students. These results demonstrate that the OHS master's program serves as a successful credential-enhancing program for dental school applicants, while also serving as a pipeline to increase the number of qualified applicants from URM groups.

B-Myb regulates the A(2B) adenosine receptor in vascular smooth muscle cells.

The A(2B) adenosine receptor (A(2B)AR) has been described to control various vascular functions, including inhibition of smooth muscle cell proliferation. Here, we sought to understand the regulation of A(2B)AR gene expression in aortic vascular smooth muscle cells (VSMCs), with a focus on the proliferation phase. Assays with A(2B)AR-beta-gal aortic VSMCs, in which the endogenous A(2B)AR gene promoter drives the expression of prokaryotic beta-galactosidase (beta-gal) instead of the endogenous A(2B)AR gene, show that beta-gal expression is upregulated when the cells are induced to exit from cell cycle arrest. Similarly, the level of A(2B)AR mRNA is upregulated in proliferating primary aortic VSMCs. In search of related mechanisms, it was noted that the A(2B)AR gene promoter contains several putative binding sites for the proliferation-inducing transcription factor, B-Myb. Using a clone of the 5' region upstream of the mouse A(2B)AR gene linked to a reporter gene, B-Myb site deletion mutants were generated. It was determined that B-Myb upregulates the A(2B)AR gene promoter, and specific promoter binding sites were identified as functional. In accordance, B-Myb also elevates endogenous A(2B)AR mRNA and receptor activity, and this activity decreases cell proliferation. Our data are novel in that they show that this proliferation-inhibiting A(2B)AR is itself an inducible receptor regulated by B-Myb.

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation.

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-alpha-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.

Collagen and major histocompatibility class II expression in mesenchymal cells from CIITA hypomorphic mice.

Major histocompatibility class II (MHC II) transactivator (CIITA) is critical for interferon-gamma (IFN-gamma)-induced repression of collagen [Xu, Y., Wang, L., Buttice, G., Sengupta, P.K., Smith, B.D., 2004. Major histocompatibility class II transactivator (CIITA) mediates repression of collagen (COL1A2) transcription by interferon gamma (IFN-gamma). J. Biol. Chem. 279, 41319-41332] and activation of MHC II transcription. To better understand the role of CIITA and IFN-gamma induced repression of collagen, mesenchymal cells (lung fibroblasts, adventitial fibroblasts, and smooth muscle cells) were isolated from a CIITA deficient mouse (C2ta(tm1Ccum)). IFN-gamma induced MHC II expression and repressed collagen type I expression in all three cell types isolated from the wild type background. As expected, IFN-gamma treatment of cells isolated from CIITA deficient mice did not induce MHC II production or activate the MHC II promoter. Interestingly, collagen gene expression and promoter activity was similar to that of wild type. Moreover, IFN-gamma induced CIITA mRNA and a truncated form of CIITA protein in all cells isolated from CIITA deficient mice. Most importantly, truncated CIITA occupied the collagen alpha 2(I) gene (col1a2) transcription start site during IFN-gamma treatment, but it did not occupy the MHC II promoter as judged by chromatin immunoprecipitation assays. Exogenous expression of a similar truncated form of CIITA maintained its ability to repress col1a2 transcription, but lost its ability to activate MHC II gene transcription suggesting a role for the CIITA C-terminal domain in activation, but not repression. IFN-gamma induced primarily types I and IV CIITA isoforms in the mouse cells. All three isoforms of CIITA were capable of repressing col1a2 and activating MHC II gene transcription. These data suggest that the previously described CIITA knockout mouse carries a hypomorphic mutation, rather than a null mutation. The removal of the leucine rich region in CIITA blocks activation of MHC II without altering repression of collagen transcription.

Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix.

Smooth muscle cells contribute to extracellular matrix remodeling during atherogenesis. De-differentiated, synthetic smooth muscle cells are involved in processes of migration, proliferation and changes in expression of extracellular matrix components, all of which contribute to loss of homeostasis accompanying atherogenesis. Elevated levels of acute phase proteins, including serum amyloid A (SAA), are associated with an increased risk for atherosclerosis. Although infection with periodontal and respiratory pathogens via activation of inflammatory cell Toll-like receptor (TLR)2 has been linked to vascular disease, little is known about smooth muscle cell TLR2 in atherosclerosis. This study addresses the role of SAA and TLR2 activation on smooth muscle cell matrix gene expression and insoluble elastin accumulation. Cultured rat aortic smooth muscle cells were treated with SAA or TLR2 agonists and the effect on expression of matrix metallopeptidase 9 (MMP9) and tropoelastin studied. SAA up-regulated MMP9 expression. Tropoelastin is an MMP9 substrate and decreased tropoelastin levels in SAA-treated cells supported the concept of extracellular matrix remodeling. Interestingly, SAA-induced down-regulation of tropoelastin was not only evident at the protein level but at the level of gene transcription as well. Contributions of proteasomes, nuclear factor κ B and CCAAT/enhancer binding protein ß on regulation of MMP9 vs. tropoleastin expression were revealed. Effects on Mmp9 and Eln mRNA expression persisted with long-term SAA treatment, resulting in decreased insoluble elastin accumulation. Interestingly, the SAA effects were TLR2-dependent and TLR2 activation by bacterial ligands also induced MMP9 expression and decreased tropoelastin expression. These data reveal a novel mechanism whereby SAA and/or infection induce changes in vascular elastin consistent with atherosclerosis.

Effects of elastase on the mechanical and failure properties of engineered elastin-rich matrices.

Pulmonary emphysema and vessel wall aneurysms are diseases characterized by elastolytic damage to elastin fibers that leads to mechanical failure. To model this, neonatal rat aortic smooth muscle cells were cultured, accumulating an extracellular matrix rich in elastin, and mechanical measurements were made before and during enzymatic digestion of elastin. Specifically, the cells in the cultures were killed with sodium azide, the cultures were lifted from the flask, cut into small strips, and fixed to a computer-controlled lever arm and a force transducer. The strips were subjected to a broadband displacement signal to study the dynamic mechanical properties of the samples. Also, quasi-static stress-strain curves were measured. The dynamic data were fit to a linear viscoelastic model to estimate the tissues' loss (G) and storage (H) modulus coefficients, which were evaluated before and during 30 min of elastase treatment, at which point a failure test was performed. G and H decreased significantly to 30% of their baseline values after 30 min. The failure stress of control samples was approximately 15 times higher than that of the digested samples. Understanding the structure-function relationship of elastin networks and the effects of elastolytic injury on their mechanical properties can lead to the elucidation of the mechanism of elastin fiber failure and evaluation of possible treatments to enhance repair in diseases involving elastolytic injury.

B-Myb represses vascular smooth muscle cell collagen gene expression and inhibits neointima formation after arterial injury.

OBJECTIVE: The function of B-Myb, a negative regulator of vascular smooth muscle cell (SMC) matrix gene transcription, was analyzed in the vasculature. METHODS AND RESULTS: Mice were generated in which the human B-myb gene was driven by the basal cytomegalovirus promoter, and 3 founders were identified. Mice appeared to develop normally, and human B-myb was expressed in the aortas. Total B-Myb levels were elevated in aortas of adult transgenic versus wild-type (WT) animals and varied inversely with alpha1(I) collagen mRNA expression. However, neonatal WT and transgenic aortas displayed comparable levels of alpha1(I) collagen mRNA, likely resulting from elevated levels of cyclin A, which ablated repression by B-Myb. Aortic SMCs from adult transgenic animals displayed decreased alpha1(I) collagen mRNA levels. To examine the role of B-Myb after vascular injury, animals were subjected to femoral artery denudation, which induces SMC-rich lesion formation. A dramatic reduction in neointima formation and lumenal narrowing was observed in arteries of B-myb transgenic versus WT mice 4 weeks after injury. CONCLUSIONS: Data indicate that B-Myb, which inhibits matrix gene expression in the adult vessel wall, reduces neointima formation after vascular injury. To analyze B-Myb function in the vasculature, mice overexpressing B-myb were generated. Neonates displayed normal alpha1(I) collagen mRNA levels, whereas adults expressed decreased collagen mRNA in aortas and isolated vascular SMCs. On femoral artery denudation, neointima formation was dramatically reduced in B-myb transgenic mice.

PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is considered to be one of the master regulators of adipocyte differentiation. PPARgamma2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPARgamma2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPARgamma2. Oil Red O staining revealed that PPARgamma2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPARgamma2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPARgamma2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis.

Analysis of gene ontology features in microarray data using the Proteome BioKnowledge Library.

Microarray technology has resulted in an explosion of complex, valuable data. Integrating data analysis tools with a comprehensive underlying database would allow efficient identification of common properties among differentially regulated genes. In this study we sought to compare the utility of various databases in microarray analysis. The Proteome BioKnowledge Library (BKL), a manually curated, proteome-wide compilation of the scientific literature, was used to generate a list of Gene Ontology (GO) Biological Process (BP) terms enriched among proteins involved in cardiovascular disease. Analysis of DNA microarray data generated in a study of rat vascular smooth muscle cell responses revealed significant enrichment in a number of GO BPs that were also enriched among cardiovascular disease-related proteins. Using annotation from LocusLink and chip annotation from the Gene Expression Omnibus yielded fewer enriched cardiovascular disease-associated GO BP terms. Data sets of orthologous genes from mouse and human were generated using the BKL Retriever. Analysis of these sets focusing on BKL Disease annotation, revealed a significant association of these genes with cardiovascular disease. These results and the extensive presence of experimental evidence for BKL GO and Disease features, underscore the benefits of using this database for microarray analysis.

B-Myb represses elastin gene expression in aortic smooth muscle cells.

B-Myb represses collagen gene transcription in vascular smooth muscle cells (SMCs) in vitro and in vivo. Here we sought to determine whether elastin is similarly repressed by B-Myb. Levels of tropoelastin mRNA and protein were lower in aortas and isolated SMCs of adult transgenic mice expressing the human B-myb gene, driven by the basal cytomegalovirus promoter, compared with age-matched wild type (WT) animals. However, the vessel wall architecture and levels of insoluble elastin revealed no differences. Since elastin deposition occurs early in development, microarray analysis was performed using nontransgenic mice. Aortic levels of tropoelastin mRNA were low during embryonal growth and increased substantially in neonates, whereas B-myb levels varied inversely. Tropoelastin mRNA expression in aortas of 6-day-old neonatal transgenic and WT animals was comparable. Recently, we demonstrated that cyclin A-Cdk2 prevents B-Myb-mediated repression of collagen promoter activity. Cyclin A2 levels were higher in neonatal versus adult WT or transgenic mouse aortas. Ectopic cyclin A expression reversed the ability of B-Myb to repress elastin gene promoter activity in adult SMCs. These results demonstrate for the first time that B-Myb represses SMC elastin gene expression and that cyclin A plays a role in the developmental regulation of elastin gene expression in the aorta. Furthermore, the findings provide additional insight into the mechanism of B-myb-mediated resistance to femoral artery injury.

Vascular smooth muscle cell polyploidization involves changes in chromosome passenger proteins and an endomitotic cell cycle.

Vascular smooth muscle cell polyploidization occurs during normal development and is enhanced under physiologic stress, but the mechanism of this cell cycle has not been explored. We show via time-lapse video imaging and immunofluorescence analyses that primary vascular smooth muscle cells (VSMC) undergo an endomitotic-type cell cycle, including a normal progression through part of mitosis. Mononuclear polyploid cells are generated by defects in sister chromatid separation and/or segregation, and cellular binucleation occurs by reversal of cytokinesis. To obtain further leads to regulators involved, we examined the chromosomal passenger proteins, Aurora B, inner centromere protein and Survivin, and concluded that Aurora B and inner centromere protein are normally colocalized in centromeres, the midzone, and the midbody during mitosis. Survivin, however, is dim and diffused; it does not colocalize with either Aurora B or inner centromere protein in VSMC, which could account for defects in sister chromatid separation and/or segregation and reversal of cytokinesis. In accordance with the reported dependency of Aurora B activity on Survivin, the Aurora B substrate, vimentin, is not phosphorylated during cytokinesis. Finally, the data show that ectopically expressed Survivin inhibits polyploidization in vascular smooth muscle cells. Hence, aberrant chromosome passenger protein activity and endomitosis are associated with VSMC polyploidization.

A(3) adenosine receptor deficiency does not influence atherogenesis.

Atherosclerosis is a multifactorial disease, the progression of which is modulated by several factors, including inflammation and hypercholesterolemia. The A(3) adenosine receptor (A(3)AR) has been reported to affect mast cell degranulation leading to inflammation, as well as to influence cardiovascular homeostasis. Here, we show that its deletion can also impact vascular smooth muscle cell (VSMC) proliferation in vitro. Based on these observations, we hypothesized that A(3)AR deficiency would affect atheromatous lesion development in vivo. Our results indicate that the expression of the matrix enzyme lysyl oxidase (LO) is increased while the proliferation potential of VSMC is decreased in A(3)AR-null aortas. This is in accordance with the previously reported inverse correlation between LO level and proliferation. Nevertheless, we found that A(3)-deficiency does not protect vessels against atherogenesis. This was demonstrated in mouse models of high fat diet-induced atherosclerosis and guidewire-induced femoral artery injury. We conclude that the contributions of the A(3)AR to inflammation and to modulating LO levels are not significant enough to control vascular response to injury.