Integrating social determinants of health principles into the preclinical medical curriculum via student-led pedagogical modalities.

BACKGROUND: Dismantling structural inequities in health care requires that physicians understand the impacts of social determinants of health (SDH). Although many medical schools incorporate SDH education, integration of these principles into the preclinical curriculum remains challenging. METHODS: Students and faculty at the University of Vermont, Larner College of Medicine developed the Social Medicine Theme of the Week (SMTW), a peer-teaching approach to integrating SDH topics across the preclinical curriculum as part of a broader social medicine curriculum. Students created objectives to link SDH-related topics to the weekly curriculum and presented them to the class. Student innovation led to the incorporation of creative online infographics that were published in the curriculum calendar. First year medical students and faculty members were surveyed to assess preferences and educational impact of the SMTW announcements with accompanying infographics. RESULTS: Of the 40 student respondents, 77.5% reported that their knowledge of SDH had improved due to the SMTW. Most students (82.5%) preferred the infographic modality over traditional teaching modalities. Faculty respondents reported limited engagement with the SMTW and, although they supported the need for these objectives, many (61%) found it difficult to integrate SDH content into their class materials. CONCLUSION: Student-led infographics are a popular method of integrating SDH content in the preclinical curriculum that can be optimized through faculty orientation and support. Success for this type of instruction requires opportunities for student developers, integration and formal assessment of objectives, faculty engagement and training, and institutional support for creating and delivering a robust social medicine curriculum.

The design and implementation of a longitudinal social medicine curriculum at the University of Vermont's Larner College of Medicine.

BACKGROUND: Despite an abundant literature advocating that social determinants of health (SDH) be taught during undergraduate medical education, there are few detailed descriptions of how to design and implement longitudinal core curricula that is delivered to all students and accomplishes this goal. METHODS: In this paper, we describe the design and implementation of a social medicine curriculum at the University of Vermont's Larner College of Medicine (UVM Larner). Using Kern's principles, we designed a longitudinal curriculum that extends through both preclinical and clinical training for all students and focused on integrating SDH material directly into basic science and clinical training. RESULTS: We successfully developed and implemented two primary tools, a "Social Medicine Theme of the Week" (SMTW) in preclinical training, and SDH rounds in the clinical setting to deliver SDH content to all learners at UVM Larner. CONCLUSIONS: Extensive student-faculty partnerships, robust needs assessment, and focusing on longitudinal and integrated SDH content delivery to all students were key features that contributed to successful design and implementation.

A new roadmap for social medicine curriculum design based on mixed methods student and faculty evaluations of the preclinical curriculum.

BACKGROUND: To support the development of social medicine curricula that empower medical school graduates to redress health inequities, we conducted a mixed methods student and faculty evaluation of an expanded and innovative preclinical social medicine curriculum. METHODS: We implemented a longitudinal, interactive preclinical social medicine curriculum that was closely integrated with foundational science teaching then conducted a survey-based mixed methods student and faculty curriculum evaluation. Based on these results, we propose a novel conceptual roadmap for social medicine curriculum design. RESULTS: Student and faculty evaluations of an expanded and innovative longitudinal preclinical social medicine curriculum were strongly favorable. Both student and faculty respondents indicated a particular desire for deeper coverage of race and poverty among other social medicine domains. Qualitative student evaluations highlighted the importance of faculty champions to social medicine teaching as well as the educational impact of stories that exemplify the practical impact of the social determinants of health on specific patient experiences. Qualitative faculty evaluations pointed to the challenges of curriculum integration and the need for faculty career development in social medicine teaching. CONCLUSIONS: Based on mixed methods student and faculty curriculum evaluation data, we propose a novel conceptual roadmap for the design of social medicine curricula at other institutions.

Induction of microvascular network growth in the mouse mesentery.

OBJECTIVE: Motivated by observations of mesenteries harvested from mice treated with tamoxifen dissolved in oil for inducible gene mutation studies, the objective of this study was to demonstrate that microvascular growth can be induced in the avascular mouse mesentery tissue. METHODS: C57BL/6 mice were administered an IP injection for five consecutive days of: saline, sunflower oil, tamoxifen dissolved in sunflower oil, corn oil, or peanut oil. RESULTS: Twenty-one days post-injection, zero tissues from saline group contained branching microvascular networks. In contrast, all tissues from the three oils and tamoxifen groups contained vascular networks with arterioles, venules, and capillaries. Smooth muscle cells and pericytes were present in their expected locations and wrapping morphologies. Significant increases in vascularized tissue area and vascular density were observed when compared to saline group, but sunflower oil and tamoxifen group were not significantly different. Vascularized tissues also contained LYVE-1-positive and Prox1-positive lymphatic networks, indicating that lymphangiogenesis was stimulated. When comparing the different oils, vascularized tissue area and vascular density of sunflower oil were significantly higher than corn and peanut oils. CONCLUSIONS: These results provide novel evidence supporting that induction of microvascular network growth into the normally avascular mouse mesentery is possible.

Assessing the effects of threonyl-tRNA synthetase on angiogenesis-related responses.

Several recent reports have found a connection between specific aminoacyl-tRNA synthetases and the regulation of angiogenesis. As this new area of research is explored, it is important to have reliable assays to assess the specific angiogenesis functions of these enzymes. This review provides information about specific in vitro and in vivo methods that were used to assess the angiogenic functions of threonyl-tRNA synthetase including endothelial cell migration and tube assays as well as chorioallantoic membrane and tumor vascularization assays. The theory and discussion include best methods of analysis and quantification along with the advantages and limitations of each type of assay.

Differential regulation of GLUT1 and GLUT8 expression by hypoxia in mammary epithelial cells.

Glucose is a major substrate for milk synthesis and is taken up from the blood by mammary epithelial cells (MECs) through facilitative glucose transporters (GLUTs). The expression levels of GLUT1 and GLUT8 are upregulated dramatically in the mammary gland from late pregnancy through early lactation stages. This study aimed to test the hypothesis that this increase in GLUT1 and GLUT8 expression involves hypoxia signaling through hypoxia inducible factor-1α (HIF-1α) in MECs. Mouse mammary glands showed significantly more hypoxia in midpregnancy through early lactation stages compared with in the virgin stage, as stained by the hypoxia marker pimonidazole HCl. Treatment with hypoxia (2% O2) significantly stimulated glucose uptake and GLUT1 mRNA and protein expression, but decreased GLUT8 mRNA expression in bovine MECs. In MECs, hypoxia also increased the levels of HIF-1α protein in the nuclei, and siRNA against HIF-1α completely abolished the hypoxia-induced upregulation of GLUT1, while having no effect on GLUT8 expression. A 5'-RCGTG-3' core HIF-1α binding sequence was identified 3.7 kb upstream of the bovine GLUT1 gene, and HIF-1α binding to this site was increased during hypoxia. In conclusion, the mammary glands in pregnant and lactating animals are hypoxic, and MECs respond to this hypoxia by increasing GLUT1 expression and glucose uptake through a HIF-1α-dependent mechanism. GLUT8 expression, however, is negatively regulated by hypoxia through a HIF-1α-independent pathway. The regulation of glucose transporters through hypoxia-mediated gene transcription in the mammary gland may provide an important physiological mechanism for MECs to meet the metabolic demands of mammary development and lactation.

Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer.

BACKGROUND: Ovarian tumors create a dynamic microenvironment that promotes angiogenesis and reduces immune responses. Our research has revealed that threonyl-tRNA synthetase (TARS) has an extracellular angiogenic activity separate from its function in protein synthesis. The objective of this study was to test the hypothesis that TARS expression in clinical samples correlates with angiogenic markers and ovarian cancer progression. METHODS: Protein and mRNA databases were explored to correlate TARS expression with ovarian cancer. Serial sections of paraffin embedded ovarian tissues from 70 patients diagnosed with epithelial ovarian cancer and 12 control patients were assessed for expression of TARS, vascular endothelial growth factor (VEGF) and PECAM using immunohistochemistry. TARS secretion from SK-OV-3 human ovarian cancer cells was measured. Serum samples from 31 tissue-matched patients were analyzed by ELISA for TARS, CA-125, and tumor necrosis factor-α (TNF-α). RESULTS: There was a strong association between the tumor expression of TARS and advancing stage of epithelial ovarian cancer (p < 0.001). TARS expression and localization were also correlated with VEGF (p < 0.001). A significant proportion of samples included heavy TARS staining of infiltrating leukocytes which also correlated with stage (p = 0.017). TARS was secreted by ovarian cancer cells, and patient serum TARS was related to tumor TARS and angiogenic markers, but did not achieve significance with respect to stage. Multivariate Cox proportional hazard models revealed a surprising inverse relationship between TARS expression and mortality risk in late stage disease (p = 0.062). CONCLUSIONS: TARS expression is increased in epithelial ovarian cancer and correlates with markers of angiogenic progression. These findings and the association of TARS with disease survival provide clinical validation that TARS is associated with angiogenesis in ovarian cancer. These results encourage further study of TARS as a regulator of the tumor microenvironment and possible target for diagnosis and/or treatment in ovarian cancer.

Regulation of angiogenesis by aminoacyl-tRNA synthetases.

In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs) have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis.

Hypoxia-mediated biological control.

When oxygen demand is greater than oxygen supply, cells need to rapidly adjust their metabolism in order for the tissue to survive. Oxygen sensing by an organism influences a host of processes including growth, development, metabolism, pH homeostasis, and angiogenesis. Hypoxia also contributes to a wide number of human diseases including vascular disease, inflammatory conditions and cancer. Recently, major advances have been made in understanding the response of cells and tissues to hypoxia with the goal of providing mechanistic insight and novel therapeutic targets. In this article we review both the normal biological effects of hypoxia as well as the alterations that occur in specific disease conditions with an emphasis on the cell signaling and gene transcription mechanisms that underlie the changes associated with chronic hypoxia. Comparisons of studies in the fields of cardiac ischemia and tumor angiogenesis reveal the complexities within the microenvironment that control responses to hypoxia. It is clear that more interaction between researchers in these fields will improve the development of therapies that either promote or prevent hypoxic responses.

Negative regulation of HIF-1α by an FBW7-mediated degradation pathway during hypoxia.

Hypoxia inducible factor-1α (HIF-1α) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF-1α is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel-Lindau; however, little is known about the regulation of HIF-1α in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3ß (GSK3ß) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF-1α phosphorylation by GSK3ß in hypoxia leads to interaction with FBW7 and ubiquitin-dependent degradation. Expression of constitutively active GSK3ß reduced HIF-1α protein and transcriptional activity and increased ubiquitination of HIF-1α in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3ß promoted HIF-1α stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF-1α stabilization when FBW7 was overexpressed and both the elevation of HIF-1α levels and decrease in ubiquitinated HIF-1α when FBW7 was suppressed. Furthermore, HIF-1α associated with FBW7γ by co-immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3ß phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK-OV-3 cell lines expressing suppressed GSK3ß or FBW7. These data introduce a new mechanism for regulation of HIF-1α during hypoxia that utilizes phosphorylation to target HIF-1α for ubiquitin-dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis.

Activation of hypoxia-inducible factor-1 protects airway epithelium against oxidant-induced barrier dysfunction.

The respiratory epithelium forms an important barrier against inhaled pollutants and microorganisms, and its barrier function is often compromised during inflammatory airway diseases. Epithelial activation of hypoxia-inducible factor-1 (HIF-1) represents one feature of airway inflammation, but the functional importance of HIF-1 within the respiratory epithelium is largely unknown. Using primary mouse tracheal epithelial (MTE) cells or immortalized human bronchial epithelial cells (16HBE14o-), we evaluated the impact of HIF-1 activation on loss of epithelial barrier function during oxidative stress. Exposure of either 16HBE14o- or MTE cells to H(2)O(2) resulted in significant loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-dextran (4 kDa), and this was attenuated significantly after prior activation of HIF-1 by preexposure to hypoxia (2% O(2); 6 h) or the hypoxia mimics CoCl(2) or dimethyloxalylglycine (DMOG). Oxidative barrier loss was associated with reduced levels of the tight junction protein occludin and with hyperoxidation of the antioxidant enzyme peroxiredoxin (Prx-SO(2)H), events that were also attenuated by prior activation of HIF-1. Involvement of HIF-1 in these protective effects was confirmed using the pharmacological inhibitor YC-1 and by short-hairpin RNA knockdown of HIF-1α. The protective effects of HIF-1 were associated with induction of sestrin-2, a hypoxia-inducible enzyme known to reduce oxidative stress and minimize Prx hyperoxidation. Together, our results suggest that loss of epithelial barrier integrity by oxidative stress is minimized by activation of HIF-1, in part by induction of sestrin-2.

A protein kinase Cdelta-dependent protein kinase D pathway modulates ERK1/2 and JNK1/2 phosphorylation and Bim-associated apoptosis by asbestos.

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cdelta-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCdelta-/- mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCdelta-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCdelta, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCdelta-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos.

Methylation-controlled J protein promotes c-Jun degradation to prevent ABCB1 transporter expression.

Methylation-controlled J protein (MCJ) is a newly identified member of the DnaJ family of cochaperones. Hypermethylation-mediated transcriptional silencing of the MCJ gene has been associated with increased chemotherapeutic resistance in ovarian cancer. However, the biology and function of MCJ remain unknown. Here we show that MCJ is a type II transmembrane cochaperone localized in the Golgi network and present only in vertebrates. MCJ is expressed in drug-sensitive breast cancer cells but not in multidrug-resistant cells. The inhibition of MCJ expression increases resistance to specific drugs by inducing expression of the ABCB1 drug transporter that prevents intracellular drug accumulation. The induction of ABCB1 gene expression is mediated by increased levels of c-Jun due to an impaired degradation of this transcription factor in the absence of MCJ. Thus, MCJ is required in these cells to prevent c-Jun-mediated expression of ABCB1 and maintain drug response.

Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells.

Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H2O2) initiates an increase in Ca2+/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 (ERK1/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1/2 and CREB in C10 cells. Application of H2O2 increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H2O2 increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H2O2-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1/2 and CREB that mediates cell survival during oxidant stress.

Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCdelta-dependent pathway in murine lung.

Asbestos is a known inflammatory, carcinogenic, and fibrotic agent, but the mechanisms leading to asbestos-induced lung diseases are unclear. Using a murine inhalation model of fibrogenesis, we show that asbestos causes significant increases in mRNA levels of lung matrix metalloproteinases (MMPs 12 and 13) and tissue inhibitor of metalloproteinases (TIMP1), as well as increased activities of MMP 2, 9, and 12 in bronchoalveolar lavage fluids (BALF). Asbestos-exposed PKCdelta knockout (PKCdelta-/-) mice exhibited decreased expression of lung MMP12 and MMP13 compared with asbestos-exposed wild-type mice. Studies using small molecule inhibitors in murine alveolar epithelial type II cells (C10) and primary lung fibroblasts confirmed that asbestos transcriptionally up-regulates MMPs via an EGFR (or other growth factor receptors)/PI3K/PKCdelta/ERK1/2 pathway. Moreover, use of a broad-spectrum MMP inhibitor showed that MMPs play an important role in further enhancing asbestos-induced signaling events by activating EGFR. These data reveal a potentially important link between asbestos signaling and integrity of the extracellular matrix (ECM) that likely contributes to asbestos-induced lung remodeling and diseases.

Store-operated Ca2+ entry activates the CREB transcription factor in vascular smooth muscle.

Ca2+-regulated gene transcription is a critical component of arterial responses to injury, hypertension, and tumor-stimulated angiogenesis. The Ca2+/cAMP response element binding protein (CREB), a transcription factor that regulates expression of many genes, is activated by Ca2+-induced phosphorylation. Multiple Ca2+ entry pathways may contribute to CREB activation in vascular smooth muscle including voltage-dependent Ca2+ channels and store-operated Ca2+ entry (SOCE). To investigate a role for SOCE in CREB activation, we measured CREB phosphorylation using immunofluorescence, intracellular Ca2+ levels using a fluorescence resonance energy transfer (FRET)-based Cameleon indicator, and c-fos transcription using RT-PCR. In this study, we report that SOCE activates CREB in both cultured smooth muscle cells and intact arteries. Depletion of intracellular Ca2+ stores with thapsigargin increased nuclear phospho-CREB levels, intracellular Ca2+ concentration, and transcription of c-fos. These effects were abolished by inhibiting SOCE through lowering extracellular Ca2+ concentration or by application of 2-aminoethoxydiphenylborate and Ni2+. Inhibition of Ca2+ influx through voltage-dependent Ca2+ channels using nimodipine partially blocked intact artery responses, but was without effect in cultured smooth muscle cells. Our findings indicate that Ca2+ entry through store-operated Ca2+ channels leads to CREB activation, suggesting that SOCE contributes to the regulation of gene expression in vascular smooth muscle.

The gamma-glutamylcysteine synthetase and glutathione regulate asbestos-induced expression of activator protein-1 family members and activity.

Asbestos fibers cause persistent increases in activator protein-1 (AP-1) family member proto-oncogenes in lung epithelial and mesothelial cells that are linked to proliferation and cell transformation. Using lung epithelial cells, the progenitor cells of lung cancers, we report that crocidolite asbestos initially depletes intracellular glutathione followed by up-regulation of both catalytic and modifier subunits of gamma-glutamylcysteine synthetase. In vivo asbestos inhalation experiments confirm increased protein levels of gamma-glutamylcysteine synthetase in mouse lungs. We also show that asbestos-induced mRNA levels of fos/jun proto-oncogenes, fra-1 transactivation, and AP-1 to DNA binding activity are glutathione-dependent. Epidermal growth factor receptor activity by asbestos is blocked by N-acetyl-l-cysteine, suggesting that it is an initial redox-activated event leading to downstream AP-1 proto-oncogene up-regulation. The overexpression of subunits of gamma-glutamylcysteine synthetase in combination completely blocked asbestos-induced up-regulation of AP-1 proto-oncogene expression. However, when overexpressed individually, the modifier subunit had more dramatic effects than the catalytic subunit. Our work shows that the glutathione-controlled redox status of the epithelial cell plays a pivotal role in asbestos-induced epidermal growth factor receptor and proto-oncogene activation as well as AP-1 activity.

Nitric oxide and reactive oxygen species exert opposing effects on the stability of hypoxia-inducible factor-1alpha (HIF-1alpha) in explants of human pial arteries.

Hypoxia induces angiogenesis, partly through stabilization of hypoxia-inducible factor-1alpha (HIF-1alpha), leading to transcription of pro-angiogenic factors. Here we examined the regulation of HIF-1alpha by hypoxia and nitric oxide (NO) in explants of human cerebrovascular smooth muscle cells. Cells were treated with NO donors under normoxic or hypoxic (2% O2) conditions, followed by analysis of HIF-1alpha protein levels. Treatment with the NO donor sodium nitroprusside reduced levels of HIF-1alpha, whereas NO donors, NOC-18 and S-nitrosoglutathione, increased HIF-1alpha levels. SIN-1, which releases both NO and superoxide (O2*-), reduced HIF-1alpha levels, suggesting that inhibitory NO donors may elicit effects through peroxynitrite (ONOO*-). O2*- generation by xanthine/xanthine oxidase also reduced HIF-1alpha levels, confirming an inhibitory role for reactive oxygen species (ROS). Furthermore, superoxide dismutase increased HIF-1alpha levels, and the NO scavenger carboxy-PTIO reversed HIF-1alpha stabilization by NO donors. Effects on HIF-1alpha levels correlated with vascular endothelial growth factor transcription but did not affect HIF-1alpha transcription, as measured by RT-PCR and luciferase-reporter assays. The results indicate that HIF-1alpha is stabilized by agents that produce NO and reduce ROS but destabilized by agents that increase ROS, including O2*- and ONOO*-. Thus we propose that the effect of NO on HIF-1alpha signaling is critically dependent on the form of NO and the physiological environment of the responding cell.