Faculty Bibliography

The progression of pancreatic oncogenesis requires immune-suppressive inflammation in cooperation with oncogenic mutations. However, the drivers of intratumoral immune tolerance are uncertain. Dectin 1 is an innate immune receptor crucial for anti-fungal immunity, but its role in sterile inflammation and oncogenesis has not been well defined. Furthermore, non-pathogen-derived ligands for dectin 1 have not been characterized. We found that dectin 1 is highly expressed on macrophages in pancreatic ductal adenocarcinoma (PDA). Dectin 1 ligation accelerated the progression of PDA in mice, whereas deletion of Clec7a-the gene encoding dectin 1-or blockade of dectin 1 downstream signaling was protective. We found that dectin 1 can ligate the lectin galectin 9 in mouse and human PDA, which results in tolerogenic macrophage programming and adaptive immune suppression. Upon disruption of the dectin 1-galectin 9 axis, CD4+ and CD8+ T cells, which are dispensable for PDA progression in hosts with an intact signaling axis, become reprogrammed into indispensable mediators of anti-tumor immunity. These data suggest that targeting dectin 1 signaling is an attractive strategy for developing an immunotherapy for PDA.

Brown adipose tissue (BAT) and beige adipose tissue combust fuels for heat production in adult humans, and so constitute an appealing target for the treatment of metabolic disorders such as obesity, diabetes and hyperlipidemia. Cold exposure can enhance energy expenditure by activating BAT, and it has been shown to improve nutrient metabolism. These therapies, however, are time consuming and uncomfortable, demonstrating the need for pharmacological interventions. Recently, lipids have been identified that are released from tissues and act locally or systemically to promote insulin sensitivity and glucose tolerance; as a class, these lipids are referred to as 'lipokines'. Because BAT is a specialized metabolic tissue that takes up and burns lipids and is linked to systemic metabolic homeostasis, we hypothesized that there might be thermogenic lipokines that activate BAT in response to cold. Here we show that the lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) is a stimulator of BAT activity, and that its levels are negatively correlated with body-mass index and insulin sensitivity. Using a global lipidomic analysis, we found that 12,13-diHOME was increased in the circulation of humans and mice exposed to cold. Furthermore, we found that the enzymes that produce 12,13-diHOME were uniquely induced in BAT by cold stimulation. The injection of 12,13-diHOME acutely activated BAT fuel uptake and enhanced cold tolerance, which resulted in decreased levels of serum triglycerides. Mechanistically, 12,13-diHOME increased fatty acid (FA) uptake into brown adipocytes by promoting the translocation of the FA transporters FATP1 and CD36 to the cell membrane. These data suggest that 12,13-diHOME, or a functional analog, could be developed as a treatment for metabolic disorders.

Background and Rationale: Mutations in Plakophilin-2 (PKP2) are the most common cause of ARVC, an inherited disease characterized by fibro- or fibrofatty infiltration of RV predominance, ventricular arrhythmias and sudden death in the young. The relation between PKP2 expression and the heart transcriptome in vivo, is unknown. Furthermore, while splicing misregulation has been associated with other inherited diseases, PKP2-dependent exon usage differences remain unexplored. We generated a murine line of cardiac-restricted, tamoxifen activated PKP2 deficiency ("PKP2-cKO") and defined PKP2- dependent exon usage in adult non-failing hearts. Methods and Results: The first disease manifestation was an increase in RV area, detected by echocardiography 14 days after tamoxifen injection (14 days post-injection or "dpi"), followed by marked RV dilation and reparative fibrosis (21 dpi), then bi-ventricular dilated cardiomyopathy (28 dpi), heart failure and death (30-50 dpi). To capture the earliest molecular events, hearts 14 dpi were used for RNAseq and exon usage. Comparing RV vs LV revealed minor changes in transcript abundance, but significant differences in alternative splicing (AS) program. We found ~75% of differentially spliced exons flanked by sequences that bind RBFox2, an RNA-binding protein that acts as central AS regulator of the adult heart, and that is necessary to maintain cardiac structure. Western blot analysis at 14 dpi and thereafter showed reduced abundance of RBFox2. RNAseq at 21 dpi showed that in addition to RBFox2, transcripts were reduced for RBFox1, MBNL1, MBNL2 and RBM20 (also molecules that control the AS program). Exon usage analysis at 21 dpi identified massive AS misregulation, similar to that of a failing heart, even though ejection fraction at this stage was ~50%. Misregulated genes included several involved in electrical rhythm and intracellular calcium homeostasis. Conclusion: We generated a model of PKP2-dependent ARVC. Our studies point to a previously unrecognized association between a desmosomal molecule, a splicing regulator, and the control of electrical and mechanical function. AS misregulation may be a substrate for sudden unexpected arrhythmic death in the young

Wound healing remains a global issue of disability, cost, and health. Addition of cells from the stromal vascular fraction (SVF) of adipose tissue has been shown to increase the rate of full thickness wound closure. This study aimed to investigate the angiogenic mechanisms of CD248+ SVF cells in the context of full thickness excisional wounds. Single cell transcriptional analysis was used to identify and cluster angiogenic gene-expressing cells, which was then correlated with surface marker expression. SVF cells isolated from human lipoaspirate were FACS sorted based on the presence of CD248. Cells were analyzed for angiogenic gene expression and ability to promote microvascular tubule formation in vitro. Following this, 6mm full thickness dermal wounds were created on the dorsa of immunocompromised mice and then treated with CD248+, CD248-, or unsorted SVF cells delivered in a pullalan-collagen hydrogel or the hydrogel alone. Wounds were measured every other day photometrically until closure. Wounds were also evaluated histologically at 7 and 14 days post-wounding and when fully healed to assess for reepithelialization and development of neovasculature. Wounds treated with CD248+ cells healed significantly faster than other treatment groups, and at 7 days, had quantitatively more reepithelialization. Concurrently, immunohistochemistry of CD31 revealed a much higher presence of vascularity in the CD248+ SVF cells treated group at the time of healing and at 14 days post-op, consistent with a pro-angiogenic effect of CD248+ cells in vivo. Therefore, using CD248+ pro-angiogenic cells obtained from SVF presents a viable strategy in wound healing by promoting increased vessel growth in the wound.

Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.

Mitochondrial connexin 43 (Cx43) plays a key role in cardiac cytoprotection caused by repeated exposure to short periods of non-lethal ischemia/reperfusion, a condition known as ischemic preconditioning. Cx43 also forms calcium (Ca2+)-permeable hemichannels that may potentially lead to mitochondrial Ca2+ overload and cell death. Here, we studied the role of Cx43 in facilitating mitochondrial Ca2+ entry and investigated its downstream consequences. To that purpose, we used various connexin-targeting peptides interacting with extracellular (Gap26) and intracellular (Gap19, RRNYRRNY) Cx43 domains, and tested their effect on mitochondrial dye- and Ca2+-uptake, electrophysiological properties of plasmalemmal and mitochondrial Cx43 channels, and cell injury/cell death. Our results in isolated mice cardiac subsarcolemmal mitochondria indicate that Cx43 forms hemichannels that contribute to Ca2+ entry and may trigger permeability transition and cell injury/death. RRNYRRNY displayed the strongest effects in all assays and inhibited plasma membrane as well as mitochondrial Cx43 hemichannels. RRNYRRNY also strongly reduced the infarct size in ex vivo cardiac ischemia-reperfusion studies. These results indicate that Cx43 contributes to mitochondrial Ca2+ homeostasis and is involved in triggering cell injury/death pathways that can be inhibited by RRNYRRNY peptide.

Delayed wound healing is a major complication of diabetes occurring in approximately 15% of chronic diabetic patients. It not only significantly affects patients' quality of life but also poses a major economic burden to the health care system. Most efforts have been focused on accelerating wound reepithelialization and closure. However, even after healing the quality of healed tissue in diabetics is abnormal and recurrence is common (50-75%). Thus, understanding how diabetes alters the ultimate mechanical properties of healed wounds will be important to develop more effective approaches for this condition. Focal adhesion kinase is an intracellular protein kinase that plays critical roles in cell migration, focal adhesion formation, and is an important component of cellular mechanotransduction. We have found that focal adhesion kinase expression is downregulated under a high glucose condition both in vitro and in vivo. This is secondary to increased activity of calpain 1, the primary enzyme responsible for focal adhesion kinase degradation, which becomes induced in hyperglycemia. We demonstrate that selective inhibition of calpain 1 activation improves wound healing and normalizes the mechanical properties of diabetic skin, suggesting a new therapeutic approach to prevent diabetic wound recurrence.

The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. In this study we show increased fatty degeneration of the bone marrow of adult ank/ank mice, which lack a functional ANK protein. In addition, isolated bone marrow stromal cells (BMSCs) isolated from ank/ank mice showed a decreased proliferation rate and osteogenic differentiation potential, and an increased adipogenic differentiation potential compared to BMSCs isolated from wild type (WT) littermates. Wnt signaling pathway PCR array analysis revealed that Wnt ligands, Wnt receptors and Wnt signaling proteins that stimulate osteoblast differentiation were expressed at markedly lower levels in ank/ank BMSCs than in WT BMSCs. Lack of ANK function also resulted in impaired bone fracture healing, as indicated by a smaller callus formed and delayed bone formation in the callus site. Whereas 5weeks after fracture, the fractured bone in WT mice was further remodeled and restored to original shape, the fractured bone in ank/ank mice was not fully restored and remodeled to original shape. In conclusion, our study provides evidence that ANK plays a critical role in the adipogenic/osteogenic fate decision of adult mesenchymal precursor cells. ANK functions in precursor cells are required for osteogenic differentiation of these cells during adult bone homeostasis and repair, whereas lack of ANK functions favors adipogenic differentiation.

OBJECTIVE: The purpose of this study was to determine whether the finding of failed fracture implants in association with lower extremity long bone fracture nonunion portends worse clinical or functional outcome following surgical nonunion repair. DESIGN: Retrospective analysis of prospectively collected data. SETTING: Academic Medical Center. PATIENTS: One hundred eighty-one patients who presented to our institution over a 10-year period and underwent surgical repair of a lower extremity fracture nonunion. INTERVENTION: Surgical repair of lower extremity fracture nonunion. MAIN OUTCOME MEASUREMENTS: Time to union, postoperative complications, VAS pain scores, and Short Musculoskeletal Function Assessment (SMFA) scores following lower extremity nonunion repair. Data was analyzed to assess for differences in postoperative outcomes based on the integrity of fracture implants at the time of nonunion diagnosis. Implant integrity was defined using 3 groups: broken implants (BI), implants intact (II), and no implants (NI). RESULTS: There was no significant difference in time to union following surgery between the BI, II, or NI groups (mean 8.1 months vs 7.6 months vs 6.2 months, respectively). Fourteen patients (7.7%) failed to heal, including 5 BI patients, 7 II patients, and 2 NI patients. One tibial nonunion patient in each of the 3 groups underwent amputation for persistent nonunion following multiple failed revision attempts at a mean of 4.8 years after initial injury. There was no difference in postoperative pain scores, the rate of postoperative complications, or functional outcome scores identified between the 3 groups. CONCLUSION: The finding of failed fracture implants at the time of lower extremity long bone nonunion diagnosis does not portend worse clinical or functional outcome following surgical nonunion repair. Patients who present with failed fracture implants at the time of nonunion diagnosis can anticipate similar time to union, complication rates, and functional outcomes when compared to patients who present with intact implants or those with history of nonoperative management. LEVEL OF EVIDENCE: Prognostic Level IV.

Homeostasis is a fundamental property of living organisms enabling the human body to withstand internal and external insults. In several chronic diseases, and especially in cancer, many homeostatic mechanisms are deranged. Pancreatic cancer in particular is notorious for its ability to invoke an intense fibroinflammatory stromal reaction facilitating its progression and resistance to treatment. In the past decade, several seminal discoveries have elucidated previously unrecognized modes of commandeering the host's defense systems. Here we review novel discoveries in pancreatic cancer immunobiology and attempt to integrate the notion of deranged homeostasis in the pathogenesis of this disease. We also highlight areas of controversy and obstacles that need to be overcome, hoping to further our mechanistic insight into this malignancy.