United States

BACKGROUND: Immunotherapy in many tumors is limited by tumor heterogeneity and low mutational burden. While the vast majority of tumor-specific antigens (neoantigens) are identified through somatic mutations, recent studies indicate aberrant RNA-splicing (neojunctions) as a new source for targets. Our spatial splicing neoantigen identification platform (SNIPP) characterizes a novel class of clonally-expressed splicing-derived neoantigens that elicit a CD8+ T-cell-mediated tumor killing response. METHODS: SNIPP identified public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) &gt; 10%) and not in GTEx normal tissue RNA-seq data (PSR &lt; 1%) across 12 cancer types. To characterize intratumorally-conserved neojunctions, we performed maximally-distanced multi-site biopsies (n=535) within glioma patients (n=56) and obtained RNA-seq data from each intratumoral site. This was additionally performed on publicly-available multi-site sequencing data from other cancer types. Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. Neoantigen-specific TCR sequences characterized from subsequent PBMC in vitro sensitization and 10x V(D)J scRNA-seq were transduced into CD8+ T-cells for immunogenicity and cytotoxicity assays against glioma cell lines. Differential gene expression (DESeq2) and gene set enrichment analyses (GSEA) is performed between tumor subtypes within cancers, and a Pearson correlation matrix is generated to identify correlated neojunctions with specific splicing factor expression. CRISPRi knockdown of select splicing factors followed by qPCR of their corresponding neojunctions are performed to determine the correlative mechanism for neojunction expression. RESULTS: Our pipeline identified 789 public neojunctions, with 32 neojunctions concurrently identified in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. IVS and subsequent 10x VDJ scRNA-seq identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in &gt; 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in various pan-cancer cell lines. Furthermore, IDH1-mutant oligodendroglioma samples demonstrated significantly elevated expression of neojunctions over IDH1-mutant astrocytoma and IDH1wt subtypes. Differential gene expression (DESeq2) identified decreased expression of splicing factors due to oligodendroglioma-specific co-deletion of Chromosomes 1p/19q. siRNA knockdown of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding neojunctions. CONCLUSION: Our unique SNIPP neoantigen discovery platform identified novel public tumor-wide splice-derived neoantigens and reactive TCRs, and its pan-cancer application characterized candidates targetable across multiple diseases.

AMA Research Challenge 2024 

Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens across cancers

BACKGROUND: Immunotherapy in many tumors is limited by tumor heterogeneity and low mutational burden. While the vast majority of tumor-specific antigens (neoantigens) are identified through somatic mutations, recent studies indicate aberrant RNA-splicing (neojunctions) as a new source for targets. Our spatial splicing neoantigen identification platform (SNIPP) characterizes a novel class of clonally-expressed splicing-derived neoantigens that elicit a CD8+ T-cell-mediated tumor killing response. METHODS: SNIPP identified public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) > 10%) and not in GTEx normal tissue RNA-seq data (PSR < 1%) across 12 cancer types. To characterize intratumorally-conserved neojunctions, we performed maximally-distanced multi-site biopsies (n=535) within glioma patients (n=56) and obtained RNA-seq data from each intratumoral site. This was additionally performed on publicly-available multi-site sequencing data from other cancer types. Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. Neoantigen-specific TCR sequences characterized from subsequent PBMC in vitro sensitization and 10x V(D)J scRNA-seq were transduced into CD8+ T-cells for immunogenicity and cytotoxicity assays against glioma cell lines. Differential gene expression (DESeq2) and gene set enrichment analyses (GSEA) is performed between tumor subtypes within cancers, and a Pearson correlation matrix is generated to identify correlated neojunctions with specific splicing factor expression. CRISPRi knockdown of select splicing factors followed by qPCR of their corresponding neojunctions are performed to determine the correlative mechanism for neojunction expression. RESULTS: Our pipeline identified 789 public neojunctions, with 32 neojunctions concurrently identified in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. IVS and subsequent 10x VDJ scRNA-seq identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in various pan-cancer cell lines. Furthermore, IDH1-mutant oligodendroglioma samples demonstrated significantly elevated expression of neojunctions over IDH1-mutant astrocytoma and IDH1wt subtypes. Differential gene expression (DESeq2) identified decreased expression of splicing factors due to oligodendroglioma-specific co-deletion of Chromosomes 1p/19q. siRNA knockdown of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding neojunctions. CONCLUSION: Our unique SNIPP neoantigen discovery platform identified novel public tumor-wide splice-derived neoantigens and reactive TCRs, and its pan-cancer application characterized candidates targetable across multiple diseases.

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Background
Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant inherited syndrome of dysregulated angiogenesis presenting with arteriovenous malformations in multiple organ systems. Despite the significant complications associated with HHT, FDA-approved therapeutic options remain unavailable. In HHT, bypassing the ACVRL1 mutation is critical to restore downstream TGF-β signaling. Extracellular vesicle (EVs) mediated delivery of recombinant proteins offer a pioneering approach, in biocompatible fashion, to treat dysregulated angiogenesis in HHT. Engineered Bone Morphogenic Protein 2 and 7 extracellular vesicles (BMP2/7-EV) may offer a potential therapeutic approach for HHT by bypassing the defective ACVRL1 and reactivating SMAD signaling.
Methods
We utilized an ex ovo chicken chorioallantoic membrane (CAM) model to investigate the effects of BMP2/7-EVs. Fertilized eggs were incubated at 37C and transferred to weigh boats on developmental day 3. On day 9, each respective treatment was applied: 50 µL of either PBS (vehicle control), ALK1-Fc (500 ng/mL), BMP2/7-EV (1.78e8 particles/ml), or a combination of BMP2/7-EV (1.78e8 particles/ml) and ALK1-Fc (500 ng/mL) onto three different areas of each embryo. Microscopy at days 9 through 11 was used to document the vessel development. IKOSA software was used to generate images quantifying branch number. Statistical significance compared to control was determined by two-way ANOVA with Tukey post hoc analysis.
Results
ALK1-Fc treatment at concentrations above 100ng/mL induced hypervascularization, demonstrated by an increase vascular branching in the native ex ovo CAM model. This effect was most pronounced at 500ng/mL. BMP2/7-EV treatment of native ex ovo CAM model and ALK1-Fc CAM model mimicking HHT behavior showed a trend towards vascular regression although the differences were not statistically significant in this pilot study. 
Conclusion
The CAM assay model demonstrates promising potential for HHT research, providing a cost-effective and ethical alternative to mammalian models, allowing direct visualization of vascular changes, and offering a platform for testing angiogenic modulators. While the BMP2/7-EV treatment showed a trend towards normalizing vessel formation in the ALK1-Fc model mimicking HHT, further optimization and repeated experiments are necessary to draw definitive conclusions. This approach not only enhances our understanding of HHT pathophysiology but also paves the way for the development of targeted therapies for HHT patients, potentially improving their quality of life. Future studies will focus on refining the model, conducting more extensive studies to ensure the reproducibility of our results and exploring how various factors influence vessel formation in our model.

Engineered BMP2/BMP7 extracellular vesicles as an anti-angiogenic strategy for Hereditary Hemorrhagic Telangiectasia Using an ex vivo Chick Chorioallantoic Membrane Model

INTRODUCTION: 
Post traumatic and chronic osteoarthritis (OA) share the underlying feature of damaged hyaline cartilage and exposure of type-II collagen (CII) in the damaged tissue due to erosive proteolytic degradation triggered by low grade ongoing inflammation. Adipose tissue derived mesenchymal stem cells (ADSC) have shown efficacy in the treatment of osteoarthritis through their ability to mitigate the host inflammatory response through paracrine signaling and possess capability for chondrogenic differentiation. However in vivo, very few ADSC bind to the damaged cartilage or differentiate in the OA lesion. Our study offers a solution to these obstacles through the creation of injectable type I collagen (Col I) microspheres loaded with ADSC and linked to an E4D4 IgG monoclonal antibody that specifically targets the type II collagen (MabCII) unmasked in osteoarthritic hyaline cartilage. Upon binding to damaged cartilage, the ADSC carried by the microsphere rapidly migrate onto the damaged cartilage surface. We propose that MabCII linked to Col I microspheres can serve as an intraarticular carrier for transport and targeting large numbers of ADSC to osteoarthritic tissue while promoting chondrogenic differentiation.

METHODS: 
Isolation and culture of ADSC: The ADSC were isolated from subcutaneous fat tissue of 3-4 months old pigs as described previously (1). All animal protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Tennessee Health Science Center. The adipose tissue was digested with 200 μg/mL collagenase II solution (Sigma) to yield ADSC that were cultured in Dulbecco's Modified Eagle Medium-High Glucose (DMEM-HG) (Gibco) supplemented with fetal bovine calf serum (FBS) and antibiotics at 37°C and 5% CO2. The ADSC were characterized by flow cytometry and for multigenic differentiation before experimental use. 

Preparation of MabCII targeted Col I scaffold: 
Lyophilized purified native Col I from fetal calf skin was the gift of Dr. David Brand, Memphis VA Medical Center. The E4D4 IgG monoclonal antibody was isolated and characterized as described previously (2). The experimental group of MabCII targeted scaffolds were constructed using a two-step EDC/NHS crosslinking reaction (ThermoFischer) that bound hydroxyl groups on Col I to primary amine groups on MabCII in a 2:3 molar ratio. The efficiency of conjugation of MabCII conjugation on the Col I scaffold was measured by protein assay of the supernatant after pelleting the collagen by centrifugation at 1000g’s for 10 minutes. Cellular scaffolds were assembled in a solution of DMEM-HG + FBS using 4mg/ml homogenized MabCII-Col I and 16 million cells/ml ADSC. 

Experimental group and analysis: 
5ul droplets of the cells (80,000 cells) in a 4 mg/ml collagen suspension were plated on a non-tissue culture petri dish to form microsphere pellets and then cultured in DMEM-HG supplemented with FBS and antibiotics at 37°C and 5% CO2 for three days. At this time point, microsphere pellets were either collected for experimentation, or left on tissue culture plate and imaged under light microscopy after 4, 6, and 12 days (Figure 1). Control groups were established using pelleted ADSC (80,000 cells/ 5 microliters) without any Col I and ADSC integrated in a Col I scaffold without linked MabCII similarly cultured. To test the migration of ADSC from the scaffold onto damaged cartilage tissue, ADSC were stained with a fluorescent dye (Vivotrack680, PerkinElmer) before pellet formation. A 5ul MabCII linked scaffolded pellet was subsequently placed on to a 5 mm trypsinized cartilage explant and imaged after 6 days using a Keyence BZ-X800 microscope with a Cy5.5 filter (NIKON). To demonstrate targeting efficiency of our MabCII linked scaffolds, microspheres of fluorescent ADSC, pelleted alone or with Col I or MabCII/Col I were suspended in a 2 ml test tube on a rocker table with 5 mm damaged (trypsinized) cartilage explants. IVIS images were obtained at 4 and 24 hours and total fluorescence of ROI was calculated by Living Image 4.2 software. 

RESULTS: 
ADSC integrated in Col I microspheres and cultured demonstrated release from microsphere and growth on tissue culture plate (Figure 1). Additionally, over 6 days in culture, ADSC migrated from their microsphere attached to the cartilage explant and spread on damaged cartilage tissue as seen by the spread of fluorescence outside of the stationary ADSC/MabCII microsphere (Figure 1). This migration was again demonstrated by scanning electron photography of stationary 5ul ADSC + Col I pellets after 4 days of incubation on trypsinized cartilage explants. The images exhibited ADSC viability on day 4 by capturing active cell division on the damaged cartilage (Figure 2). Finally, IVIS imaging of quantified fluorescence showed targeting of only ADSC/Col I/ MabCII microspheres to damaged cartilage at 4 hours, while the explants incubated alone or with ADSC or ADSC/Col I exhibited no florescence at this time point (Figure 3). ADSC/Col I linked MabCII pellets remained fluorescent on damaged cartilage at 24 hours. 

DISCUSSION: 
Adipose tissue derived mesenchymal stem cell regenerative therapy for regrowth of damaged cartilage for osteoarthritis relies on ADSC’s ability to differentiate into chondrocytes on damaged hyaline cartilage tissue. We demonstrate in vitro that the use of a CII targeting MabCII linked to a Col I scaffold induced chondrogenesis of ADSC and increased the specificity and rapidity whereby ADSC can be delivered to osteoarthritic tissue. Our targeted pellets not only bound more rapidly to damaged cartilage, but they also remained attached after 24hrs and migrated onto the damaged cartilage. These results show promise for an in vivo study using our MabCII targeted Col I microspheres as an intra-articular injectable therapeutic treatment for osteoarthritis. 

SIGNIFICANCE: 
The creation of an injectable cellular scaffold with ability to target ADSC to type-II collagen on damaged hyaline cartilage increases the localization of mesenchymal stem cells to an osteoarthritic lesion at early stages. This increases ADSC therapeutic potential as a treatment for OA.

REFERENCES: 
1. Bhatti F, Kim SJ, Yi AK, Hasty K.A., Cho. H. “Cytoprotective Role of Vitamin E in Porcine Adipose-Tissue Derived Mesenchymal Stem Cells against Hydrogen-peroxide Induced Oxidative Stress” Cell and Tissue Research. 2018, 374(1):111-120. (PMID: 29951700). 2. Bedingfield SK, Colazo JM, Yu F, Liu DD, Jackson MA, Himmel LE, Cho H, Crofford LJ, Hasty KA, Duvall CL. Amelioration of post-traumatic osteoarthritis via nanoparticle depots delivering small interfering RNA to damaged cartilage. Nat Biomed Eng. 2021 Sep;5(9):1069-1083. (PMID: 34413494).

Mesenchymal Stem Cell Therapy Targeted to Damaged OA CartilageUsing Type I Collagen Microspheres Linked to Monoclonal Antibody to Type II Collagen

G0/G1 Switch 2 regulates radiation sensitivity of human head and neck cancer through a G1-lipid checkpoint

Patel, N., Kalen, A. L., Asha, W., Son, J., Schibler, J., Goswami, P. C., and Sarsour, E. H.

Background: Head and neck squamous cell carcinoma (HNSCC) resistance to radiotherapy has prompted a need to develop adaptive radiation therapy protocols to improve patient outcomes. Previous studies showed that HNSCC tumors with more G0+G1 phase cells (low proliferative index, LPI) are resistant to radiation versus HNSCC tumors with more S+G2 phase cells (high proliferative index, HPI). RNA-seq and bioinformatics have identified lipid metabolism as the major intrinsic pathway that differs between HPI and LPI HNSCC cultures. mRNA and protein levels of G0/G1 Switch 2 gene (G0S2), regulator of quiescence and lipid metabolism, have been found to be upregulated in LPI compared to HPI HNSCC cultures. G0S2 negatively regulates adipose triglyceride lipase (ATGL), resulting in less lipolytic activity. This study investigates the hypothesis that lipid metabolism regulates cell cycle phase specific radiation sensitivity of HNSCC cells.

Methods: In vitro cell culture methods were optimized within individual human tongue squamous cell carcinoma cell lines to obtain cultures with high proliferative index (HPI; >60% S+G2+M; higher RNA content) and cultures with low proliferative index (LPI; >70% G0+G1 cells; lower RNA content). Flow cytometry was utilised to measure DNA and RNA content. To override G0S2 action, we incubated LPI cultures with the fatty acid palmitate and examined cellular metabolic stress markers, such as reactive oxygen species and lipid peroxidation.

Results: LPI cultures were noted to be more radiation resistant compared to HPI cultures. Radiation sensitivity of HPI cultures was found to be associated with a significant increase in lipid peroxidation. Compared to controls, LPI cultures treated with palmitate showed increased reactive oxygen species levels, lipid peroxidation and oxygen consumption rate coupled with increased mitochondrial fission. Palmitate also resulted in significant radiation sensitization. Furthermore, using the fluorescent based cell cycle real-time imaging system, we showed that palmitate treatment sustained cell proliferation (higher S+G2) compared to controls (higher G1).

Conclusion: In summary, we demonstrated that G0S2 dependent lipid metabolism regulates cell cycle phase specific radiation sensitivity of HNSCC cells. G0S2 served as an effective inhibitor of lipolysis, which in turn reduced availability of free fatty acids and increased the radiation resistance of cancer cells. siG0S2 treatment of LPI cultures recruited cells into the proliferative cycle and exacerbated radiation sensitivity. We identified G0S2 and free fatty acids as novel targets for radiation therapy.

G0-G1 Switch 2 regulates G1 -lipid checkpoint dependent radiation sensitivity of human head and neck cancer cells

Background
Prostate Cancer (PCa) is the leading cause of cancer cases in U.S. men and the second leading cause of male cancer death. If detected in its initial stages, PCa is curable with surgery or radiation, or may even be amenable to periodic surveillance. However, an advanced form the disease, termed castration-resistant PCa (CRPC), is not curable, and accounts for most PCa mortality. CRPC is defined as a form of PCa that continues progressing despite using androgen deprivation therapy (ADT), the standard of care for advanced PCa. A western diet – rich in lipids- has been shown to increase the incidence of PCa, as well as the risk of advanced PCa. However, the exact mechanism of these outcomes remains to be elucidated. We hypothesize that dietary lipids impact the susceptibility of CRPC to ferroptosis, a lipid peroxidation-dependent form of cell death. Lipid peroxidation is dependent on the PUFA content in the cell membrane.

Methods
To study this hypothesis, we have used western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), mass spectrometry, crystal violet assays, flow cytometry approaches, and RNA sequencing.


Results
We have determined that the PUFA content is elevated in CRPC models relative to a model of castration-sensitive PCa (CSPC). The CRPC models with elevated PUFA content also exhibit increased sensitivity to ferroptosis inducing agents. To explore the link between diet and ferroptosis in PCa, we cultured these models in lipid depleted (LD) medium and identified a non-apoptotic increase in dead cells. This same treatment led to an increased level of lipid peroxides. Upon culture in LD medium, the hallmark cholesterol homeostasis gene set, including genes involved in cholesterol synthesis, was the top enriched gene set across all models. Mass spectrometry-based metabolomics also showed a significant decrease in cholesteryl ester levels across all models cultured in LD medium. Interestingly, we observed that CRPC models, but not the CSPC model, showed dependence on extracellular cholesterol for their growth.

Conclusion
These results reveal an increased susceptibility of CRPC models to ferroptosis and a role for dietary lipids, specifically cholesterol, in the modulation of this mechanism of cell death. This finding can be used to design trials focused on further understanding the combination of ferroptosis inducing agents with dietary modifications for the treatment of CRPC.


Elucidating the Role of Extracellular Lipids in Ferroptosis in Castration Resistant Prostate Cancer

Background:
Placental inflammation, defined as exposure to uncontrolled inflammation before birth, is prevalent in preterm birth (PTB) making up 10-15% of all births in the United States; it is a major cause of neonatal morbidity/mortality. Unfortunately, there are currently no available therapies targeting placental inflammation. Thus, there is great need to understand the cell-specific mechanism of prenatal inflammation at the placenta for the development of targeted therapeutics. 
Methods:
Retrospective clinical chart review was completed on 228 patients who delivered either Term or PTB (≥40 or <37 weeks gestation respectively) at Mayo Clinic Rochester. Paraffin-embedded placentas were obtained for analysis of immune cells (i.e. Hofbauer cells - HBCs), by imaging mass cytometry (IMC) and immunohistochemistry (IHC). Fresh biopsies of placenta and cell suspensions of cytotrophoblasts (CTBs) and HBCs from uncomplicated term pregnancies were exposed to inflammatory inducers (lipopolysaccharide [LPS] and polyI:C [PIC]) with production/secretion of inflammatory cytokines measured by ELISA and flow cytometry. One-way ANOVA, chi-squared, or Fisher’s exact test were conducted as appropriate.
Results:
Representative patients were identified based on covariates from in-depth chart review with equal number of male and female fetuses. To assess what immune cells are in the placenta, IMC analysis on representative patients found 22 distinct cell populations including CD163+CD68+ HBCs as the only immune cell detected within placenta villi (with some T cells and monocytes detected in the maternal space only) and a higher cell diversity in PTB compared to Term samples. To assess any differences in the number of HBCs in the placenta, IHC analysis on the whole cohort found significantly elevated number of CD68+CD163+ HBCs in placentas from PTB, regardless of labor classification, vs. Term. In explant models, exposure to LPS and PIC significantly increased the secretion/production of pro-inflammatory cytokines (IL-1β, IL-6, MCP-1, TNFα). Cell suspensions experiments revealed that HBCs had significantly more IL-6 production compared to CTBs at basal levels. In LPS-stimulated conditions, IL-6 production was significantly increased in HBCs only, compared to the lack of significant upregulation in CTBs.
Conclusion:
PTB groups had increased number of HBCs compared to term groups regardless of labor classification, suggesting that labor itself does not drive the placental immune profile. Spatial analysis in placenta confirmed HBCs as the only immune cell. Placental analysis by explants and cell suspensions determined HBCs as the driver of the observed inflammatory profile. Future work will investigate interactions between HBCs and trophoblasts and their spatially resolved roles in prenatal inflammation.

Hofbauer cells: drivers of the inflammatory profile at the placenta

Pediatric high-grade gliomas (pHGGs) are some of the most aggressive brain tumors, responsible for over 40% of all childhood tumor-related deaths. Although the molecular landscape of pHGG has been characterized, pHGG lack effective molecular targeted therapies. The epidermal growth factor receptor (EGFR), a common oncogenic driver in cancer, is amplified or mutated in two out of four specific subsets of pHGGs: those characterized by histone 3 (H3) and isocitrate dehydrogenase (IDH) wild-type status plus diffuse midline gliomas, particularly when bithalamic. However, it has previously been unknown whether aberrant EGFR can be successfully targeted pharmacologically to help treat pHGGs.

In the first study of its kind, we tested the anti-tumor activity of ERAS-801, a highly brain-penetrant EGFR inhibitor developed by our laboratory in a patient-derived orthotopic xenograft mouse model of H3/IDH wild-type pHGG with EGFR amplification. Excitingly, we observed that mice treated with ERAS-801 demonstrated both dramatically slower tumor growth and >100% longer survival (median survival of over 60 days) compared to vehicle-treated mice (median survival less than 25 days). Furthermore, treatment with ERAS-801 affords better tumor control than radiation, and combining ERAS-801 with radiation demonstrates a synergistic effect on tumor control and mouse survival.

These preliminary data have led us to hypothesize that EGFR inhibition predominantly slows down the growth of pHGGs with EGFR alterations and that the exquisite sensitivity seen of pHGGs in response to ERAS-801 stems from high apoptotic priming (i.e., proximity to the apoptotic cliff). We intend to use ERAS-801 as a meaningful treatment strategy for H3/IDH wild-type pHGGs and diffuse midline gliomas with EGFR alterations. Strong preliminary data indicate a role for EGFR inhibition by ERAS-801 for treating pHGGs that harbor EGFR amplification or mutations, thus serving as both an adjunct to radiation or as an alternative treatment option entirely. Accordingly, our work can allow patients to avoid the neurotoxicity of radiation at a young age and even provide those who are too young for radiation with life-prolonging therapy.

A novel brain-penetrant tyrosine kinase inhibitor for pediatric high-grade gliomas

Abstract Title: Dapagliflozin attenuates loss of Lrp2 to preserve circulating apolipoprotein M and endothelial integrity

Background
Sodium-glucose co-transporter inhibitors (SGLT2i) have been found to reduce inflammation and preserve vascular integrity, although no unifying mechanism explains the link between the renal effects of SGLT2i and endothelial barrier function. Apolipoprotein M (ApoM), a lipocalin associated with high-density lipoprotein, is inversely associated with mortality in inflammatory conditions and is critical for maintenance of vascular integrity via sphingosine-1-phosphate (S1P) signaling. We set out to test the hypothesis that pre-treatment with selective SGLT2i dapagliflozin (Dapa) improves vascular integrity in endotoxin (lipopolysaccharide, LPS)-treated mice via the ApoM/S1P pathway. Dapa has well-described clinical benefits in type 2 diabetes mellitus (T2DM), heart failure (HF) and chronic kidney disease.

Methods
To test our hypothesis, mice with diet-induced obesity were gavaged with vehicle or Dapa for 4 days prior to LPS (10 mg/kg IP). Mice receiving Dapa restored circulating ApoM levels by increasing proximal tubular Lrp2 compared with vehicle in LPS-treated mice. Proximal tubule-specific knockout of the multi-ligand protein receptor Lrp2 blocked the effect of Dapa on circulating ApoM. In vitro, Dapa stimulated uptake of GFP-ApoM in a Lrp2-dependent manner. In LPS models utilizing ApoM transgenic mice, knockout mice, and S1P receptor 1 and 3 inhibitors, ApoM/S1P signaling was necessary and sufficient for the beneficial effects of Dapa in the LPS model.

Results
In the setting of acute inflammation, Dapa maintains levels of Lrp2, leading to preservation of ApoM, which in turn promotes endothelial barrier integrity and improves hemodynamics.

Conclusion
Our studies suggest a novel mechanism by which Dapa pre-treatment reduces LPS-induced endothelial leak in an ApoM/S1P-dependent manner and can preserve intravascular volume in the acute inflammatory setting. Dapa preserved cardiac preload, as evidenced by preserved end-diastolic volume index, coronary sinus area, and had an increased right atrial contraction pressure during a severe inflammatory reaction, all of which led to the beneficial effects of Dapa in acute inflammation.

Dapagliflozin attenuates loss of Lrp2 to preserve circulating apolipoprotein M and endothelial integrity

Introduction
Distal radius fractures are becoming increasing prevalent in our aging population. Locked volar plating remains the preferred surgical treatment. Prior studies investigating optimal locking plate constructs for fracture stability have predominantly used extra-articular fracture models. Many surgeons have adopted unicortical distal fixation to minimize risk of extensor tendon injury. This study aimed to determine the biomechanical effect of unicortical distal locking screws and pegs on stability in comminuted, intra-articular distal radius fracture cadavers. 

Methods
We applied volar-locking distal radius plates to 24 cadaveric radiuses. The radiuses were divided into 4 groups based on distal locking fixation: 100% length bicortical screws, 75% length screws, 100% length pegs, and 75% length pegs. Osteotomies simulated AO type 23 C2 dorsally comminuted, intra-articular fractures. We determined each construct’s stiffness under physiologic loads (axial compression, dorsal bending, and volar bending) before and after 1,000 cycles of axial conditioning and before axial load to clinical failure (2mm of displacement) and catastrophic failure. We used Analysis of Covariance (ANCOVA) to compare differences of means between groups and Tukey Honest Significant Difference (HSD) test for post-hoc analysis. 

Results 
There were no significant differences in age, weight, or bone mineral density (BMD) between groups. Stiffness to volar bending and dorsal bending were statistically equivalent between groups before and after cycling loading. Final stiffness under axial load was statistically equivalent for all groups. Force to clinical failure was statistically equivalent between 100% screw length (308N) and 75% screw length (351N) constructs, and equivalent between 100% peg length (127N) and 75% peg length (150N) constructs. Forces to clinical failure were significantly greater for 100% and 75% screw lengths compared to their peg counterparts (p=0.0253 and p=0.0125 respectively). Force to clinical failure was significantly greater for 75% screw lengths than 100% smooth pegs (p=0.005). Force to catastrophic failure was statistically equivalent between all groups. 

Conclusions 
We recommend the use of 75% length screws or pegs over their bicortical counterparts to prevent dorsal cortex penetration without compromising stability. Surgeons may consider using threaded locking screws over smooth pegs in treating more complex intra-articular distal radius fractures. 



Investigating the Effect of Distal Screw and Peg Length on Stability in Volar Plating of Comminuted, Intra-articular Distal Radius Fractures

Background
The incidence of acute sport injuries in children is on the rise, with 2.6 annual million emergency room visits for sports-related injury. Concurrently, opioid prescribing practices have increased in recent decades, especially in pediatric populations. To help curb opioid usage, a multimodal pain management guideline should be instituted. Further investigation is needed on the precision of regional anesthetic in performing an adductor canal nerve block (ACB) in pediatric knee arthroscopy procedures. Variations in nerve locations within the thigh may impact postoperative pain and opioid usage. We are interested in studying the optimal location of ACB placement to reduce postoperative pain and consequently reduce opioid medication usage after pediatric knee arthroscopy.

Methods
A prospective, double-blinded randomized control trial consisting of 80 pediatric patients (ages 5-17) undergoing knee arthroscopy at St. Christopher’s Hospital for Children, approved by the Institutional Review Board. Enrolled patients are randomized to receive an ultrasound-guided placement of an ACB using 0.2% ropivacaine in one of three locations in the thigh: proximal, middle, distal, or control group with saline. The primary data collected are postoperative pain scores and amount of opioids taken, standardized to morphine milligram equivalents (MME), in the 24 and 48-hour postoperative period. Secondary outcomes included correlational assessment of pre and postoperative anxiety of patients and caregivers, correlation between patient anxiety and pain, and a postoperative dermatome sensation test. Data analysis performed with ANOVA test. Complete results including analysis of primary and secondary outcomes are shown in Tables 1 and 2.

Results
60 patients (mean age 15.5) have completed this actively enrolling study. This includes 14 in the proximal placement group, 15 in the middle placement group, and 16 in the distal placement group, and 15 in the control group. Primary outcomes analysis showed no significant difference in pain scores or opioids taken at 24 and 48-hours postoperatively among groups. Increased anxiety positively correlated with heightened postoperative pain.

Conclusion
ACB placement in proximal, middle, and distal thigh locations provided comparable 24 and 48-hour postoperative analgesia and opioid medication usage in our first 60 patients.

Adductor Canal Regional Anesthetic Placement in Pediatric Patients for Knee Arthroscopy: Defining the Optimal Location and Analysis of Other Trends

Background
Recent evidence has demonstrated that current hypertension (HTN) diagnosis using seated blood pressure (BP) measurements alone may omit supine HTN, a condition linked with mortality. Higher resting supine systolic BP (SBP) and maximal exercise SBP are associated with increased morbidity and mortality risk, but their relationship remains unexplored in cohorts including both men and women. This study aimed to assess potential sex differences in the change from resting supine BP to maximal exercise BP to predict all-cause mortality.

Methods
This sample included 2,269 apparently healthy, nonclinical adults (1176 men, 1093 women, mean age: 45.2 +- 13.0 yr) from the Ball State Adult Fitness Longitudinal Lifestyle Study (BALL ST) cohort. Participants underwent a maximal cardiopulmonary treadmill exercise test with resting supine and peak exercise SBP recorded. Descriptive statistics summarized participant characteristics including risk factors, ethnicity, and clinical measurements like baseline supine BP and changes to peak BP. Baseline differences among men and women were assessed using unpaired t-tests. Participants were categorized into four groups based on resting and peak SBP: supine HTN (SBP >140mmHg) with exaggerated peak BP (SBP >210mmHg for men, SBP >190mmHg for women), supine HTN with normal peak BP, normal supine BP with exaggerated peak BP, and normal supine BP with normal peak BP. Kaplan-Meier survival curve analysis with Bonferroni adjustment compared these groups.

Results
Women exhibited lower resting supine SBP (124.4 +- 15.5 vs 132.3 +- 14.6 mmHg), peak SBP (166.8 +- 21.7 vs 185.3 +- 22.2 mmHg), and change from supine to peak SBP (42.4 +- 16.6 vs 53.0 +- 19.1 mmHg) compared to men (P<0.05, all). Participants were followed for an average of 19.0 +- 9.4 yr for all-cause mortality, during which 238 died (163 men, 75 women). Kaplan-Meier analysis in the whole cohort revealed lower survival in the supine HTN, normal peak SBP and supine HTN, exaggerated peak SBP groups compared with normal supine, normal peak group (P<0.05, both). Sex-specific survival curve analyses demonstrated lower survival rates for the HTN, exaggerated peak SBP compared to the normal supine, normal peak group for men and women (P<0.05, both).

Conclusion
These findings demonstrate higher all-cause mortality in both men and women with supine HTN and exaggerated peak BP responses, compared to those with normal supine SBP and normal peak responses. This highlights the importance of screening for supine HTN and incorporating resting supine BP measurements with exercise treadmill stress tests to better predict all-cause mortality.

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