Faculty Bibliography

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. Here, we demonstrate that E₅C, a recombinant protein block polymer, consisting of five repeats of elastin like polypeptide (E) and a coiled-coil domain of cartilage oligomeric matrix protein (C), is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E₅C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin, provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E₅C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E₅C gels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the progranulin-derivative Atsttrin, for therapeutic application in OA.

AIMS/OBJECTIVE:Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall and anti-inflammatory treatment strategies are currently pursued to lower cardiovascular disease burden. Modulation of recently discovered inactive rhomboid protein 2 (iRhom2) attenuates shedding of tumor necrosis factor-alpha (TNF-α) selectively from immune cells. The present study aims at investigating the impact of iRhom2 deficiency on the development of atherosclerosis. METHODS AND RESULTS/RESULTS:Low-density lipoprotein receptor (LDLR)-deficient mice with additional deficiency of iRhom2 (LDLR-/-iRhom2-/-) and control (LDLR-/-) mice were fed a Western type diet (WD) for 8 or 20 weeks to induce early or advanced atherosclerosis. Deficiency of iRhom2 resulted in a significant decrease in the size of early atherosclerotic plaques as determined in aortic root cross sections. LDLR-/-iRhom2-/- mice exhibited significantly lower serum levels of TNF-α and lower circulating and hepatic levels of cholesterol and triglycerides compared to LDLR-/- mice at 8 weeks of WD. Analyses of hepatic bile acid concentration and gene expression at 8 weeks of WD revealed that iRhom2 deficiency prevented WD-induced repression of hepatic bile acid synthesis in LDLR-/- mice. In contrast, at 20 weeks of WD plaque size, plaque composition, and serum levels of TNF-α or cholesterol were not different between genotypes. CONCLUSIONS:Modulation of inflammation by iRhom2 deficiency attenuated diet induced hyperlipidemia and early atherogenesis in LDLR-/- mice. iRhom2 deficiency did not affect diet- induced plaque burden and composition in advanced atherosclerosis in LDLR-/- mice. TRANSLATIONAL PERSPECTIVE/UNASSIGNED:iRhom2 attenuates shedding of TNF-α selectively from immune cells and therefore has emerged as a potential new target for the treatment of inflammatory diseases. In the present study, we identified iRhom2 as a critical link between inflammation, lipid metabolism, and atherogenesis. Selective iRhom2 inhibition represents a potential treatment strategy to modify atherosclerosis, particularly in the presence of enhanced inflammation as observed with diabetes mellitus or rheumatoid arthritis.

Tissue injury leads to the well-orchestrated mobilization of systemic and local innate and adaptive immune cells. During aging, immune cell recruitment is dysregulated, resulting in an aberrant inflammatory response that is detrimental for successful healing. Here, we precisely define the systemic and local immune cell response after femur fracture in young and aging mice and identify increased toll-like receptor signaling as a potential culprit for the abnormal immune cell recruitment observed in aging animals. Myd88, an upstream regulator of TLR-signaling lies at the core of this aging phenotype, and local treatment of femur fractures with a Myd88 antagonist in middle-aged mice reverses the aging phenotype of impaired fracture healing, thus offering a promising therapeutic target that could overcome the negative impact of aging on bone regeneration.

Neurodegenerative diseases are debilitating impairments that affect millions of people worldwide and are characterized by progressive degeneration of structure and function of the central or peripheral nervous system. Effective biomarkers for neurodegenerative diseases can be used to improve the diagnostic workup in the clinic as well as facilitate the development of effective disease-modifying therapies. Progranulin (PGRN) has been reported to be involved in various neurodegenerative disorders. Hence, in the current study we systematically compared the inflammation and accumulation of typical neurodegenerative disease markers in the brain tissue between PGRN knockout (PGRN KO) and wildtype (WT) mice. We found that PGRN deficiency led to significant neuron loss as well as activation of microglia and astrocytes in aged mice. Several characteristic neurodegenerative markers, including α-synuclein, TAR DNA-binding protein 43 (TDP-43), Tau, and β-amyloid, were all accumulated in the brain of PGRN-deficient mice as compared to WT mice. Moreover, higher aggregation of lipofuscin was observed in the brain tissue of PGRN-deficient mice compared with WT mice. In addition, the autophagy was also defective in the brain of PGRN-deficient mice, indicated by the abnormal expression level of autophagy marker LC3-II. Collectively, comprehensive assays support the idea that PGRN plays an important role during the development of neurodegenerative disease, indicating that PGRN might be a useful biomarker for neurodegenerative diseases in clinical settings.

The purpose of this review is to discuss the transforming growth factor beta (TGFB) binding proteins (LTBP) with respect to their participation in the activity of TGFB. We first describe pertinent aspects of the biology and cell function of the LTBPs. We then summarize the physiological consequences of LTBP loss in humans and mice. Finally, we consider a number of outstanding questions relating to LTBP function.

Transforming growth factor β (TGFβ) is a pleiotropic cytokine that plays critical roles to define cancer cell phenotypes, construct the tumor microenvironment, and suppress anti-tumor immune responses. As such, TGFβ is a lynchpin for integrating cancer cell intrinsic pathways and communication among host cells in the tumor and beyond that together affect responses to genotoxic, targeted, and immune therapy. Despite decades of preclinical and clinical studies, evidence of clinical benefit from targeting TGFβ in cancer remains elusive. Here, we review the mechanisms by which TGFβ acts to oppose successful cancer therapy, the reported prognostic and predictive value of TGFβ biomarkers, and the potential impact of inhibiting TGFβ in precision oncology. Paradoxically, the diverse mechanisms by which TGFβ impedes therapeutic response are a principal barrier to implementing TGFβ inhibitors because it is unclear which TGFβ mechanism is functional in which patient. Companion diagnostic tools and specific biomarkers of TGFβ targeted biology will be the key to exploiting TGFβ biology for patient benefit.

Barth syndrome is a multisystem disorder caused by an abnormal metabolism of the mitochondrial lipid cardiolipin. In this review, we discuss physical properties, biosynthesis, membrane assembly, and function of cardiolipin. We hypothesize that cardiolipin reduces packing stress in the inner mitochondrial membrane, which arises as a result of protein crowding. According to this hypothesis, patients with Barth syndrome are unable to meet peak energy demands because they fail to concentrate the proteins of oxidative phosphorylation to a high surface density in the inner mitochondrial membrane.

Unlike other checkpoint inhibitors, our targeted immunotherapeutic localizes to any solid tumor and simultaneouslyshields an agent of immuno suppression while presenting a signal for immunostimulation. Phosphatidylserine (PS)exposure on the extracellular surface of living tumor cells and their vasculatures provides one avenue by which thetumor microenvironment promotes immunosuppression. Extracellular surface PS is inherent to a tumor and itsvasculature, even for inoperable tumors, and its expression cannot be mutated nor affected by acquired drugresistance. Annexin A5 (AnxA5) is a direct, high-affinity PS-binding protein that localizes to cells with PS exposed onthe outer plasma membrane. In our studies, we conjugated a proprietary modified AnxA5, lacking cellularinternalization, to TNFalpha (AnxA5 -TNFalpha) to convert the immunosuppresive environs of a murine 4T1 triplenegative breast cancer (TNBC) into an immunostimulated one. This strategy localized the immune response to the tumor and minimized side effects, as evidenced by a lack of toxicity for up to 7 days in non-tumor bearing Balb/cfemale mice given up to 1 mg/kg. Proper assembly and functionality of AnxA5 -TNFalpha was verified simultaneouslyby ellipsometry, an optical technique similar to plasmon resonance. Fully assembled constructs were tested forbinding to PS coated slides. The degree of light polarization is proportional to the amount of PS bound by the AnxA5complex. Samples could be further incubated with TNF receptors to verify TNFalpha activity. Based on dose escalationstudies in 4T1 tumor-bearing mice where the TNBC tumors were grown in the mammary fat pads, optimal dosages were determined for AnxA5 -TNFalpha (18 mug) and AnxA5 alone as a control (180 mug). These doses were furthertested in a 4T1 growth inhibition study. Tumor size was tracked by caliper in two groups of mice (n=5/group)receiving drug treatment on days 12, 14 and 16 and a repeated measures ANOVA was conducted onmeasurements taken before, during and post-treatment. While median tumor size did not differ between control and drug treatment groups during the pre-treatment interval (p=0.84), there was a significant difference post-treatment(p<0.001) with mice receiving AnxA5 -TNFalpha having much smaller TNBC tumors. Tumors from the study were embedded in paraffin, sectioned (5 mum) and the overall immune cell content determined by H&E staining. Once it was evident there was a greater quantity of immune cells in AnxA5 -TNFalpha treated tumors vs. controls, sections were stained with validated antibodies to identify and count the immunoactivated T-cells, NK-cells and macrophages. There was a 3X greater mean percentage of CD8 and CD4 T-cells in mice receiving drug vs. control(p=0.03) along with 2.5X and 5X increases in NK-cells and M1 immunoactive macrophages, respectively.
Conclusion(s): Our AnxA5 -TNFalpha inhibits the PS inhibitor while simultaneously activating TNF activators!