Faculty Bibliography

Selective serotonin reuptake inhibitors (SSRIs) are one of the most commonly prescribed antidepressants worldwide and recent data show significant impairment of fracture healing after treatment with the SSRI fluoxetine in mice. Here, we provide evidence that the negative effects of SSRIs can be overcome by administration of the beta-blocker propranolol at the time of fracture. First, in vitro experiments established that propranolol does not affect osteogenic differentiation. We then used a murine model of intramembranous ossification to study the potential rescue effect of propranolol on SSRI-induced impaired fracture healing. MicroCT analysis revealed that fluoxetine treatment resulted in a smaller bony regenerate and that this decrease in bone formation can be overcome by co-treatment with propranolol. We then tested this in a clinically relevant model of endochondral ossification. Fluoxetine-treated mice with a femur fracture were treated with propranolol initiated at the time of fracture, and a battery of analyses demonstrated a reversal of the detrimental effect of fluoxetine on fracture healing in response to propranolol treatment. These experiments show for the first time that the negative effects of SSRIs on fracture healing can be overcome by co-treatment with a beta-blocker. This article is protected by copyright. All rights reserved.

Modeling age-related macular degeneration (AMD) is challenging, because it is a multifactorial disease. To focus on interactions between the retinal pigment epithelium (RPE) and Bruch's membrane, we generated RPE from AMD patients and used an altered extracellular matrix (ECM) that models aged Bruch's membrane. Induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from AMD patients or age-matched (normal) controls. RPE derived from iPSCs were analyzed by morphology, marker expression, transepithelial electrical resistance (TER), and phagocytosis of rod photoreceptor outer segments. Cell attachment and viability was tested on nitrite-modified ECM, a typical modification of aged Bruch's membrane. DNA microarrays with hierarchical clustering and analysis of mitochondrial function were used to elucidate possible mechanisms for the observed phenotypes. Differentiated RPE displayed cell-specific morphology and markers. The TER and phagocytic capacity were similar among iPSC-derived RPE cultures. However, distinct clusters were found for the transcriptomes of AMD and control iPSC-derived RPE. AMD-derived iPSC-RPE downregulated genes responsible for metabolic-related pathways and cell attachment. AMD-derived iPSC-RPE exhibited reduced mitochondrial respiration and ability to attach and survive on nitrite-modified ECM. Cells that did attach induced the expression of complement genes. Despite reprogramming, iPSC derived from AMD patients yielded RPE with a transcriptome that is distinct from that of age-matched controls. When challenged with an AMD-like modification of Bruch's membrane, AMD-derived iPSC-RPE activated the complement immune system.

Some cells discard undesired inherited components in bulk by forming large compartments that are subsequently eliminated. Caenorhabditis elegans primordial germ cells (PGCs) jettison mitochondria and cytoplasm by forming a large lobe that is cannibalized by intestinal cells. Although PGCs are nonmitotic, we find that lobe formation is driven by constriction of a contractile ring and requires the RhoGEF ECT-2, a RhoA activator also essential for cytokinesis. Whereas centralspindlin activates ECT-2 to promote cytokinetic contractile ring formation, we show that the ECT-2 regulator NOP-1, but not centralspindlin, is essential for PGC lobe formation. We propose that lobe contractile ring formation is locally inhibited by the PGC nucleus, which migrates to one side of the cell before the cytokinetic ring assembles on the opposite cortex. Our findings reveal how components of the cytokinetic contractile ring are reemployed during interphase to create compartments used for cellular remodeling, and they reveal differences in the spatial cues that dictate where the contractile ring will form.

During mammalian development, gonadotropin-releasing-hormone-1 neurons (GnRH-1ns) migrate from the developing vomeronasal organ (VNO) into the brain asserting control of pubertal onset and fertility. Recent data suggest that correct development of the olfactory ensheathing cells (OEC) is imperative for normal GnRH-1 neuronal migration. However, the full ensemble of molecular pathways that regulate OEC development remains to be fully deciphered. Loss-of-function of the transcription factor Gli3 is known to disrupt olfactory development, however, if Gli3 plays a role in GnRH-1 neuronal development is unclear. By analyzing Gli3 extra-toe mutants (Gli3^Xt/Xt), we found that Gli3 loss-of-function compromises the onset of achaete-scute family bHLH transcription factor 1 (Ascl-1) positive vomeronasal progenitors and the formation of OEC in the nasal mucosa. Surprisingly, GnRH-1 neurogenesis was intact in Gli3^Xt/Xt mice but they displayed significant defects in GnRH-1 neuronal migration. In contrast, Ascl-1^null mutants showed reduced neurogenesis for both vomeronasal and GnRH-1ns but less severe defects in OEC development. These observations suggest that Gli3 is critical for OEC development in the nasal mucosa and subsequent GnRH-1 neuronal migration. However, the non-overlapping phenotypes between Ascl-1 and Gli3 mutants indicate that Ascl-1, while crucial for GnRH-1 neurogenesis, is not required for normal OEC development. Since Kallmann syndrome (KS) is characterized by abnormal GnRH-1ns migration, we examined whole exome sequencing data from KS subjects. We identified and validated a GLI3 loss-of-function variant in a KS individual. These findings provide new insights into GnRH-1 and OECs development and demonstrate that human GLI3 mutations contribute to KS etiology.Significance statement. The transcription factor Gli3 is necessary for correct development of the olfactory system. However, if Gli3 plays a role in controlling GnRH-1 neuronal development has not been addressed. We found that Gli3 loss-of-function compromises the onset of Ascl-1+ vomeronasal progenitors, formation of olfactory ensheathing cells in the nasal mucosa, and impairs GnRH-1 neuronal migration to the brain. By analyzing Ascl-1^null mutants we dissociated the neurogenic defects observed in Gli3 mutants from lack of olfactory ensheathing cells in the nasal mucosa, moreover, we discovered that Ascl-1 is necessary for GnRH-1 ontogeny. Analyzing human whole exome sequencing data, we identified a GLI3 loss-of-function variant in a KS individual. Our data suggest that GLI3 is a candidate gene contributing to KS etiology.

Because the incidence of breast cancer increases decades after ionizing radiation exposure, aging has been implicated in the evolution of the tumor microenvironment and tumor progression. Here, we investigated radiation-induced carcinogenesis using a model in which the mammary glands of 10-month-old BALB/c mice were transplanted with Trp53-null mammary tissue three days after exposure to low doses of sparsely ionizing γ-radiation or densely ionizing particle radiation. Mammary transplants in aged irradiated hosts gave rise to significantly more tumors that grew more rapidly than those in sham-irradiated mice, with the most pronounced effects seen in mice irradiated with densely ionizing particle radiation. Tumor transcriptomes identified a characteristic immune signature of these aggressive cancers. Consistent with this, fast-growing tumors exhibited an immunosuppressive tumor microenvironment with few infiltrating lymphocytes, abundant immunosuppressive myeloid cells, and high cyclooxgenase-2 and TGFβ. Only aged irradiated hosts gave rise to tumors lacking cytotoxic CD8+ lymphocytes (defined here as immune desert), which also occurred in younger mice. These data suggested that host irradiation may promote immunosuppression. To test this, young chimera mice were fed chow containing a honeybee-derived compound with anti-inflammatory and immunomodulatory properties, caffeic acid phenethyl ester (CAPE). CAPE prevented the detrimental effects of host irradiation on tumor growth rate, immune signature, and immunosuppression. These data indicated that low-dose radiation, particularly densely ionizing radiation, promoted more aggressive cancers by suppressing antitumor immunity. Dietary intervention with a non-toxic immunomodulatory agent could prevent systemic effects of radiation that fuel carcinogenesis, supporting the potential of this strategy for cancer prevention.

The purpose of this investigation was to determine the role of extracellular vesicles (EVs), released from articular chondrocytes in a physiological or pathological state, in cell-cell communication with other articular chondrocytes or chondrocytic precursor cells. Conditioned medium from interleukin-1beta (IL-1β)-treated human articular chondrocytes stimulated catabolic events and inhibited type II collagen expression in articular chondrocytes to a much greater degree than medium from IL-1β-treated chondrocytes after complete removal of EVs. Vehicle-treated and IL-1β-treated human articular chondrocytes released EVs of similar size; however the number of EVs released by IL-1β-treated chondrocytes was markedly higher than the number of EVs released from vehicle-treated cells. Furthermore, our findings demonstrate that similar to medium from IL-1β-treated chondrocytes containing EVs, EVs isolated from medium of IL-1β-treated chondrocytes stimulated catabolic events in articular chondrocytes, whereas EVs isolated from the medium of vehicle-treated chondrocytes inhibited catabolic events and increased mRNA levels of aggrecan and type II collagen in IL-1β-treated chondrocytes. Furthermore, medium containing EVs from vehicle-treated articular chondrocytes or EVs isolated from this medium stimulated chondrogenesis of C3H10T1/2 cells, whereas medium containing EVs from IL-1β-treated chondrocytes or EVs isolated from this medium inhibited chondrogenesis. Our findings suggest that EVs released by articular chondrocytes play a key role in the communication between joint cells and ultimately in joint homeostasis, maintenance, pathology, and repair. This article is protected by copyright. All rights reserved.

Despite substantial progress in lung cancer immunotherapy, the overall response rate in KRAS-mutant lung adenocarcinoma (ADC) patients remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of anti-tumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused sgRNA library, and performed an in vivo CRISPR screen in a KrasG12D/P53-/- (KP) lung ADC model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T cell activation in combination with anti-PD-1. Our results provide rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy.

The role of IL-17 in cancer remains controversial. Emerging evidence suggests that during early oncogenesis IL-17 supports tumor growth, whereas in established tumors IL-17 production by γδ and Th17 cells potentiates antitumor immunity. Consequently, γδ and Th17 cells are attractive targets for immunotherapy in the IL-17 immune axis. To optimize IL-17-based immunotherapy, a deeper understanding of the cytokines dictating IL-17 production and the polarity of γδ and Th17 cells is critical. Here, we delve into the dichotomous roles of IL-17 in cancer and provide insight into the tumor microenvironment conducive for successful IL-17-based γδ and Th17 cell immunotherapy.

OBJECTIVE:To determine the prevalence of Barth syndrome in the pediatric population. STUDY DESIGN/METHODS:Data were collected from the Barth Syndrome Foundation Registry and relevant literature. With the advent of genetic testing and whole-exome sequencing, a multipronged Bayesian analysis was used to estimate the prevalence of Barth syndrome based on published data on the incidence and prevalence of cardiomyopathy and neutropenia, and the respective subpopulations of patients with Barth syndrome indicated in these publications. RESULTS:Based on 7 published studies of cardiomyopathy and 2 published studies of neutropenia, the estimated prevalence of Barth syndrome is approximately 1 case per million male population. This contrasts with 99 cases in the Barth Syndrome Foundation Registry, 58 of which indicate a US location, and only 230-250 cases known worldwide. CONCLUSIONS:It appears that Barth syndrome is greatly underdiagnosed. There is a need for better education and awareness of this rare disease to move toward early diagnosis and treatment.

BACKGROUND:The purpose of this study was to compare narcotic use in the 90-day postoperative period across orthopaedic trauma, spine, and adult reconstruction patients and examine whether patient-reported pain scores at discharge correlate with narcotic use during the 90-day postoperative period. METHODS:Electronic medical record query was done between 2012 and 2015 using diagnosis-related groups for spine, adult reconstruction, and trauma procedures. Demographics, length of stay (LOS), visual analog scale pain scores during hospitalization, and narcotics prescribed in the 90-day postoperative period were collected. Multivariate analysis and linear regression were done. RESULTS:Five thousand thirty patients were analyzed. Spine patients had the longest LOS, highest mean pain during LOS, and were prescribed the most morphine in the 90-day postoperative period. Linear regression revealed that pain scores at discharge markedly influence the quantity of narcotics prescribed in the 90-day postoperative period. DISCUSSION/CONCLUSIONS:Patient-reported pain at hospital discharge was associated with increased narcotic use in the 90-day postoperative period.