Uncovering lipid dynamics in Staphylococcus aureus osteomyelitis using multimodal imaging mass spectrometry.

Osteomyelitis occurs when Staphylococcus aureus invades the bone microenvironment, resulting in a bone marrow abscess with a spatially defined architecture of cells and biomolecules. Imaging mass spectrometry and microscopy are tools that can be employed to interrogate the lipidome of S. aureus-infected murine femurs and reveal metabolic and signaling consequences of infection. Here, nearly 250 lipids were spatially mapped to healthy and infection-associated morphological features throughout the femur, establishing composition profiles for tissue types. Ether lipids and arachidonoyl lipids were altered between cells and tissue structures in abscesses, suggesting their roles in abscess formation and inflammatory signaling. Sterols, triglycerides, bis(monoacylglycero)phosphates, and gangliosides possessed ring-like distributions throughout the abscess, suggesting a hypothesized dysregulation of lipid metabolism in a population of cells that cannot be discerned with traditional microscopy. These data provide insight into the signaling function and metabolism of cells in the fibrotic border of abscesses, likely characteristic of lipid-laden macrophages.

STING-Activating Polymer-Drug Conjugates for Cancer Immunotherapy.

The stimulator of interferon genes (STING) pathway links innate and adaptive antitumor immunity and therefore plays an important role in cancer immune surveillance. This has prompted widespread development of STING agonists for cancer immunotherapy, but pharmacological barriers continue to limit the clinical impact of STING agonists and motivate the development of drug delivery systems to improve their efficacy and/or safety. We developed SAPCon, a STING-activating polymer-drug conjugate platform based on strain-promoted azide-alkyne cycloaddition of a novel dimeric amidobenzimidazole (diABZI) STING prodrug to hydrophilic poly(dimethylacrylamide-co-azido-ethylmethacrylate) polymer chains through a cathepsin B-responsive linker to increase circulation time and enable passive tumor accumulation. We found that intravenously administered SAPCon accumulated at tumor sites, where it was endocytosed by tumor-associated myeloid cells, resulting in increased STING activation in the tumor tissue. Consequently, SAPCon promoted an immunogenic tumor microenvironment characterized by increased frequency of activated macrophages and dendritic cells and improved infiltration of CD8+ T cells, resulting in inhibition of tumor growth, prolonged survival, and enhanced response to anti-PD-1 immune checkpoint blockade in orthotopic breast cancer models. Collectively, these studies position SAPCon as a modular and programmable platform for improving the efficacy of systemically administered STING agonists for cancer immunotherapy.

The highly metastatic 4T1 breast carcinoma model possesses features of a hybrid epithelial/mesenchymal phenotype.

Epithelial-mesenchymal transitions (EMTs) are thought to promote metastasis via downregulation of E-cadherin (also known as Cdh1) and upregulation of mesenchymal markers such as N-cadherin (Cdh2) and vimentin (Vim). Contrary to this, E-cadherin is retained in many invasive carcinomas and promotes collective cell invasion. To investigate how E-cadherin regulates metastasis, we examined the highly metastatic, E-cadherin-positive murine 4T1 breast cancer model, together with the less metastatic, 4T1-related cell lines 4T07, 168FARN and 67NR. We found that 4T1 cells display a hybrid epithelial/mesenchymal phenotype with co-expression of epithelial and mesenchymal markers, whereas 4T07, 168FARN, and 67NR cells display progressively more mesenchymal phenotypes in vitro that relate inversely to their metastatic capacity in vivo. Using RNA interference and constitutive expression, we demonstrate that the expression level of E-cadherin does not determine 4T1 or 4T07 cell metastatic capacity in mice. Mechanistically, 4T1 cells possess highly dynamic, unstable cell-cell junctions and can undergo collective invasion without E-cadherin downregulation. However, 4T1 orthotopic tumors in vivo also contain subregions of EMT-like loss of E-cadherin. Thus, 4T1 cells function as a model for carcinomas with a hybrid epithelial/mesenchymal phenotype that promotes invasion and metastasis.

MALDI TIMS IMS Reveals Ganglioside Molecular Diversity within Murine S. aureus Kidney Tissue Abscesses.

Gangliosides play important roles in innate and adaptive immunity. The high degree of structural heterogeneity results in significant variability in ganglioside expression patterns and greatly complicates linking structure and function. Structural characterization at the site of infection is essential in elucidating host ganglioside function in response to invading pathogens, such as Staphylococcus aureus (S. aureus). Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables high-specificity spatial investigation of intact gangliosides. Here, ganglioside structural and spatial heterogeneity within an S. aureus-infected mouse kidney abscess was characterized. Differences in spatial distributions were observed for gangliosides of different classes and those that differ in ceramide chain composition and oligosaccharide-bound sialic acid. Furthermore, integrating trapped ion mobility spectrometry (TIMS) allowed for the gas-phase separation and visualization of monosialylated ganglioside isomers that differ in sialic acid type and position. The isomers differ in spatial distributions within the host-pathogen interface, where molecular patterns revealed new molecular zones in the abscess previously unidentified by traditional histology.

Isolation of human antibodies against influenza B neuraminidase and mechanisms of protection at the airway interface.

Influenza B viruses (IBVs) comprise a substantial portion of the circulating seasonal human influenza viruses. Here, we describe the isolation of human monoclonal antibodies (mAbs) that recognized the IBV neuraminidase (NA) glycoprotein from an individual following seasonal vaccination. Competition-binding experiments suggested the antibodies recognized two major antigenic sites. One group, which included mAb FluB-393, broadly inhibited IBV NA sialidase activity, protected prophylactically in vivo, and bound to the lateral corner of NA. The second group contained an active site mAb, FluB-400, that broadly inhibited IBV NA sialidase activity and virus replication in vitro in primary human respiratory epithelial cell cultures and protected against IBV in vivo when administered systemically or intranasally. Overall, the findings described here shape our mechanistic understanding of the human immune response to the IBV NA glycoprotein through the demonstration of two mAb delivery routes for protection against IBV and the identification of potential IBV therapeutic candidates.

Epithelial IL-2 is critical for NK cell-mediated cancer immunosurveillance in mammary glands.

Interleukin 2 (IL-2) is the first identified cytokine and its interaction with receptors has been known to shape the immune responses in many lymphoid or non-lymphoid tissues for more than four decades. Active T cells are the primary cellular source for IL-2 production and epithelial cells have never been considered the major cellular source of IL-2 under physiological conditions. It is, however, tempting to speculate that epithelial cells could potentially express IL-2 that regulates the intricate interactions between epithelial cells and lymphocytes. Datamining our recently published single-cell RNAseq in the mouse mammary gland identified IL-2 expression in mammary epithelial cells, which is induced by prolactin via the STAT5 signaling pathway. Furthermore, epithelial IL-2 plays a crucial role in maintaining the physiological functions of natural killer (NK) cells within the mammary glands. IL-2 deletion in the mammary epithelial cells leads to a significant reduction in the number and function of NK cells, which in turn results in defective immunosurveillance, expansion of luminal epithelial cells, and tumor development. Interestingly, T cells in the mammary glands are not changed, indicating the specific regulation of NK cells by epithelial IL-2 production. In agreement, we also found that human epithelial cells express IL-2 and NK cells express the highest level of IL2RB among all the immune cells. Here, we provide the first evidence that epithelial cells produce IL-2, which is critical for maintaining the physiological functions of NK cells in immunosurveillance.

In utero and postnatal ivacaftor/lumacaftor therapy rescues multiorgan disease in CFTR-F508del ferrets.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with F508del being the most prevalent mutation. The combination of CFTR modulators (potentiator and correctors) has provided benefit to CF patients carrying the F508del mutation; however, the safety and effectiveness of in utero combination modulator therapy remains unclear. We created a F508del ferret model to test whether ivacaftor/lumacaftor (VX-770/VX-809) therapy can rescue in utero and postnatal pathologies associated with CF. Using primary intestinal organoids and air-liquid interface cultures of airway epithelia, we demonstrate that the F508del mutation in ferret CFTR results in a severe folding and trafficking defect, which can be partially restored by treatment with CFTR modulators. In utero treatment of pregnant jills with ivacaftor/lumacaftor prevented meconium ileus at birth in F508del kits and sustained postnatal treatment of CF offspring improved survival and partially protected from pancreatic insufficiency. Withdrawal of ivacaftor/lumacaftor treatment from juvenile CF ferrets reestablished pancreatic and lung diseases, with altered pulmonary mechanics. These findings suggest that in utero intervention with a combination of CFTR modulators may provide therapeutic benefits to individuals with F508del. This CFTR-F508del ferret model may be useful for testing therapies using clinically translatable endpoints.

Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

Determination of Postmortem Interval in Mice.

Despite the major use of mice in biomedical research, little information is available with regard to identifying their postmortem changes and using that information to determine the postmortem interval (PMI), defined as the time after death. Both PMI and environmental conditions influence decomposition (autolysis and putrefaction) and other postmortem changes. Severe decomposition compromises lesion interpretation and disease detection and wastes limited pathology resources. The goal of this study was to assess postmortem changes in mice in room temperature cage conditions and under refrigeration at 4 °C to develop gross criteria for the potential value of further gross and histologic evaluation. We used 108 experimentally naïve C57BL/6 mice that were humanely euthanized and then allocated them into 2 experimental groups for evaluation of postmortem change: room temperature (20 to 22 °C) or refrigeration (4 °C). PMI assessments, including gross changes and histologic scoring, were performed at hours 0, 4, 8, and 12 and on days 1 to 14. Factors such as temperature, humidity, ammonia in the cage, and weight change were also documented. Our data indicates that carcasses held at room temperature decomposed faster than refrigerated carcasses. For most tissues, decomposition was evident by 12 h at room temperature as compared with 5 d under refrigeration. At room temperature, gross changes were present by day 2 as compared with day 7 under refrigeration. Mice at room temperature lost 0.78% of their baseline body weight per day as compared with 0.06% for refrigerated mice (95% CI for difference 0.67% to 0.76%, P < 0.0005). This study supports the consideration of temperature and PMI as important factors affecting the suitability of postmortem tissues for gross and histologic evaluation and indicates that storage of carcasses under refrigeration will significantly slow autolysis.

Deficiency of the lipid flippase ATP10A causes diet-induced dyslipidemia in female mice.

Genetic association studies have linked ATP10A and closely related type IV P-type ATPases (P4-ATPases) to insulin resistance and vascular complications, such as atherosclerosis. ATP10A translocates phosphatidylcholine and glucosylceramide across cell membranes, and these lipids or their metabolites play important roles in signal transduction pathways regulating metabolism. However, the influence of ATP10A on lipid metabolism in mice has not been explored. Here, we generated gene-specific Atp10A knockout mice and show that Atp10A-/- mice fed a high-fat diet did not gain excess weight relative to wild-type littermates. However, Atp10A-/- mice displayed female-specific dyslipidemia characterized by elevated plasma triglycerides, free fatty acids and cholesterol, as well as altered VLDL and HDL properties. We also observed increased circulating levels of several sphingolipid species along with reduced levels of eicosanoids and bile acids. The Atp10A-/- mice also displayed hepatic insulin resistance without perturbations to whole-body glucose homeostasis. Thus, ATP10A has a sex-specific role in regulating plasma lipid composition and maintaining hepatic liver insulin sensitivity in mice.

Tumor nano-lysate activates dendritic cells to evoke a preventative immune response.

A tumor nano-lysate "TNL" vaccine comprised of sonicated 4T1 cells was developed, characterized and implemented for the prevention of triple-negative breast cancer. This study aimed to gain a better understanding of the immune response behind the success of the vaccine in vivo, through use of ex vivo and in vivo assays. Here, we analyze the activation of various immune cells isolated from healthy mouse spleens and find that antigen-presenting cells (APCs) such as dendritic cells (DCs) are being activated following 24 h incubation with 1:10 mg TNL/mg splenocytes. These cells were further explored to determine the pathway by which activation is occurring, and it was observed that TNL are phagocytosed by DCs to activate NF-kB and c-Fos pathways, resulting in enhanced cytokine release after 24 h. An in vivo temporal analysis was performed in mice to understand the immune response at 1, 3, 7 and 10 days after one 100 µL dose of TNL consisting of 105 sonicated 4T1 cells via cardiac puncture and splenocyte and peripheral blood mononuclear cell (PBMC) analysis. Changes were observed for up to one week. A multiple dose study was performed comparing mice that were vaccinated with one dose of TNL administered every ten days for 3 doses total, as well as a PBS vehicle control. Survival for TNL-vaccinated mice was enhanced compared to the PBS control, and there was an average delay of 10 days in the onset of metastasis. The differences between the groups at the end of the study demonstrate the potential for TNL as a preventative therapeutic.

Polyomavirus-associated Disseminated T-cell Lymphoma in a Colony of Zebra Finches (Taeniopygia guttata).

Four zebra finches in a closed research colony presented with variable clinical signs, including masses, skin lesions, shivering, and/or ruffled feathers. These birds were not responsive to treatment efforts; 3 died and one was euthanized. All 4 were submitted for necropsy to determine the cause of the clinical signs. Gross necropsy and histopathologic findings from all birds resulted in a diagnosis of round cell neoplasia in multiple organs, including the skin, liver, kidney, and reproductive tract, with intranuclear inclusion bodies in the neoplastic cells. In all 4 cases, immunohistochemical staining showed strong immunoreactivity for CD3 in 70% to 80% of the neoplastic round cells, with a relatively small subset that were immunopositive for Pax5. These findings supported a diagnosis of T-cell lymphoma. Frozen liver tissue from one case was submitted for next-generation sequencing (NGS), which revealed viral RNA with 100% sequence homology to canary polyomavirus strain 34639 that had originally been identified in a European goldfinch. Formalin-fixed paraffin-embedded scrolls from another case were also submitted for NGS, which revealed viral RNA with 97.2% sequence homology to canary polyomavirus strain 37273 that had originally been identified in a canary. To localize the virus in situ, RNAscope hybridization was performed using a probe designed to target the VP1 gene of the sequenced virus in frozen liver tissue. In all 4 cases, disseminated and robust hybridization signals were detected in neoplastic cells. These findings indicate that polyomaviruses have the potential to be oncogenic in zebra finches.

Mitochondrial-targeted antioxidant attenuates preeclampsia-like phenotypes induced by syncytiotrophoblast-specific Gαq signaling.

Syncytiotrophoblast stress is theorized to drive development of preeclampsia, but its molecular causes and consequences remain largely undefined. Multiple hormones implicated in preeclampsia signal via the Gαq cascade, leading to the hypothesis that excess Gαq signaling within the syncytiotrophoblast may contribute. First, we present data supporting increased Gαq signaling and antioxidant responses within villous and syncytiotrophoblast samples of human preeclamptic placenta. Second, Gαq was activated in mouse placenta using Cre-lox and DREADD methodologies. Syncytiotrophoblast-restricted Gαq activation caused hypertension, kidney damage, proteinuria, elevated circulating proinflammatory factors, decreased placental vascularization, diminished spiral artery diameter, and augmented responses to mitochondrial-derived superoxide. Administration of the mitochondrial-targeted antioxidant Mitoquinone attenuated maternal proteinuria, lowered circulating inflammatory and anti-angiogenic mediators, and maintained placental vascularization. These data demonstrate a causal relationship between syncytiotrophoblast stress and the development of preeclampsia and identify elevated Gαq signaling and mitochondrial reactive oxygen species as a cause of this stress.

In situ lipidomics of Staphylococcus aureus osteomyelitis using imaging mass spectrometry.

Osteomyelitis occurs when Staphylococcus aureus invades the bone microenvironment, resulting in a bone marrow abscess with a spatially defined architecture of cells and biomolecules. Imaging mass spectrometry and microscopy are invaluable tools that can be employed to interrogate the lipidome of S. aureus-infected murine femurs to reveal metabolic and signaling consequences of infection. Here, nearly 250 lipids were spatially mapped to healthy and infection-associated morphological features throughout the femur, establishing composition profiles for tissue types. Ether lipids and arachidonoyl lipids were significantly altered between cells and tissue structures in abscesses, suggesting their roles in abscess formation and inflammatory signaling. Sterols, triglycerides, bis(monoacylglycero)phosphates, and gangliosides possessed ring-like distributions throughout the abscess, indicating dysregulated lipid metabolism in a subpopulation of leukocytes that cannot be discerned with traditional microscopy. These data provide chemical insight into the signaling function and metabolism of cells in the fibrotic border of abscesses, likely characteristic of lipid-laden macrophages.

Clostridioides difficile ferrosome organelles combat nutritional immunity.

Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.

Growth factor free, peptide-functionalized gelatin hydrogel promotes arteriogenesis and attenuates tissue damage in a murine model of critical limb ischemia.

Critical limb ischemia (CLI) occurs when blood flow is restricted through the arteries, resulting in ulcers, necrosis, and chronic wounds in the downstream extremities. The development of collateral arterioles (i.e. arteriogenesis), either by remodeling of pre-existing vascular networks or de novo growth of new vessels, can prevent or reverse ischemic damage, but it remains challenging to stimulate collateral arteriole development in a therapeutic context. Here, we show that a gelatin-based hydrogel, devoid of growth factors or encapsulated cells, promotes arteriogenesis and attenuates tissue damage in a murine CLI model. The gelatin hydrogel is functionalized with a peptide derived from the extracellular epitope of Type 1 cadherins. Mechanistically, these "GelCad" hydrogels promote arteriogenesis by recruiting smooth muscle cells to vessel structures in both ex vivo and in vivo assays. In a murine femoral artery ligation model of CLI, delivery of in situ crosslinking GelCad hydrogels was sufficient to restore limb perfusion and maintain tissue health for 14 days, whereas mice treated with gelatin hydrogels had extensive necrosis and autoamputated within 7 days. A small cohort of mice receiving the GelCad hydrogels were aged out to 5 months and exhibited no decline in tissue quality, indicating durability of the collateral arteriole networks. Overall, given the simplicity and off-the-shelf format of the GelCad hydrogel platform, we suggest it could have utility for CLI treatment and potentially other indications that would benefit from arteriole development.

Pharmacokinetics of Extended-release Buprenorphine in Mongolian Gerbils (Meriones unguiculatus).

Both the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act and Regulations require animals in research to receive adequate analgesia unless an exception can be scientifically justified and IACUC approved. Extended- release buprenorphine (BUP-XR) is a pharmaceutical-grade formulation that is FDA-indexed for use in mice and rats. However, this new formulation has not been evaluated in adult Mongolian gerbils (Meriones unguiculatus). Our goal was to determine whether the extrapolated dose (1 mg/kg SC) would achieve plasma buprenorphine concentrations above the murine therapeutic threshold (> 1.0 ng/mL) in male and female gerbils. We hypothesized that BUP-XR administered at 1 mg/kg would achieve the murine therapeutic threshold in both male and female gerbils until at least 48 h after injection. Gerbils received one injection of BUP-XR (1 mg/kg SC) and underwent 4 serial blood collections (0.5, 1, 2, and 4, or 0.5, 24, 48, and 72 h after injection). The average plasma buprenorphine concentrations were above 1 ng/mL within 30 min of administration for both males and females. Plasma buprenorphine concentrations remained above 1.0 ng/mL for 48 h after administration. In males, plasma buprenorphine concentrations were significantly higher at 1 h after injection as compared with females; no other significant differences were observed between sexes. Mild to moderate injection-site granulomas were observed in five of nine gerbils, presumably due to the lipid matrix of the BUP-XR formulation. Our findings demonstrate that a single BUP-XR dose (1 mg/kg SC) achieves plasma buprenorphine levels that remain above the murine therapeutic threshold of 1.0 ng/mL for up to 48 h in both sexes.

Deficiency of the lipid flippase ATP10A causes diet-induced dyslipidemia in female mice.

Genetic association studies have linked ATP10A and closely related type IV P-type ATPases (P4-ATPases) to insulin resistance and vascular complications, such as atherosclerosis. ATP10A translocates phosphatidylcholine and glucosylceramide across cell membranes, and these lipids or their metabolites play important roles in signal transduction pathways regulating metabolism. However, the influence of ATP10A on lipid metabolism in mice has not been explored. Here, we generated gene-specific Atp10A knockout mice and show that Atp10A-/- mice fed a high-fat diet did not gain excess weight relative to wild-type littermates. However, Atp10A-/- mice displayed female-specific dyslipidemia characterized by elevated plasma triglycerides, free fatty acids and cholesterol, as well as altered VLDL and HDL properties. We also observed increased circulating levels of several sphingolipid species along with reduced levels of eicosanoids and bile acids. The Atp10A-/- mice also displayed hepatic insulin resistance without perturbations to whole-body glucose homeostasis. Thus, ATP10A has a sex-specific role in regulating plasma lipid composition and maintaining hepatic liver insulin sensitivity in mice.

Albumin-binding RNAi Conjugate for Carrier Free Treatment of Arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are joint diseases that are associated with pain and lost quality of life. No disease modifying OA drugs are currently available. RA treatments are better established but are not always effective and can cause immune suppression. Here, an MMP13-selective siRNA conjugate was developed that, when delivered intravenously, docks onto endogenous albumin and promotes preferential accumulation in articular cartilage and synovia of OA and RA joints. MMP13 expression was diminished upon intravenous delivery of MMP13 siRNA conjugates, consequently decreasing multiple histological and molecular markers of disease severity, while also reducing clinical manifestations such as swelling (RA) and joint pressure sensitivity (RA and OA). Importantly, MMP13 silencing provided more comprehensive OA treatment efficacy than standard of care (steroids) or experimental MMP inhibitors. These data demonstrate the utility of albumin 'hitchhiking' for drug delivery to arthritic joints, and establish the therapeutic utility of systemically delivered anti-MMP13 siRNA conjugates in OA and RA. Editorial summary: Lipophilic siRNA conjugates optimized for albumin binding and "hitchhiking" can be leveraged to achieve preferential delivery to and gene silencing activity within arthritic joints. Chemical stabilization of the lipophilic siRNA enables intravenous siRNA delivery without lipid or polymer encapsulation. Using siRNA sequences targeting MMP13, a key driver of arthritis-related inflammation, albumin hitchhiking siRNA diminished MMP13, inflammation, and manifestations of osteoarthritis and rheumatoid arthritis at molecular, histological, and clinical levels, consistently outperforming clinical standards of care and small molecule MMP antagonists.

Growth factor-free, peptide-functionalized gelatin hydrogel promotes arteriogenesis and attenuates tissue damage in a murine model of critical limb ischemia.

Critical limb ischemia (CLI) occurs when blood flow is restricted through the arteries, resulting in ulcers, necrosis, and chronic wounds in the downstream extremities. The development of collateral arterioles (i.e. arteriogenesis), either by remodeling of pre-existing vascular networks or de novo growth of new vessels, can prevent or reverse ischemic damage, but it remains challenging to stimulate collateral arteriole development in a therapeutic context. Here, we show that a gelatin-based hydrogel, devoid of growth factors or encapsulated cells, promotes arteriogenesis and attenuates tissue damage in a murine CLI model. The gelatin hydrogel is functionalized with a peptide derived from the extracellular epitope of Type 1 cadherins. Mechanistically, these "GelCad" hydrogels promote arteriogenesis by recruiting smooth muscle cells to vessel structures in both ex vivo and in vivo assays. In a murine femoral artery ligation model of CLI, delivery of in situ crosslinking GelCad hydrogels was sufficient to restore limb perfusion and maintain tissue health for 14 days, whereas mice treated with gelatin hydrogels had extensive necrosis and autoamputated within 7 days. A small cohort of mice receiving the GelCad hydrogels were aged out to 5 months and exhibited no decline in tissue quality, indicating durability of the collateral arteriole networks. Overall, given the simplicity and off-the-shelf format of the GelCad hydrogel platform, we suggest it could have utility for CLI treatment and potentially other indications that would benefit from arteriole development.