Early Resveratrol Treatment Mitigates Joint Degeneration and Dampens Pain in a Mouse Model of Pseudoachondroplasia (PSACH).

Pseudoachondroplasia (PSACH), a severe dwarfing condition associated with early-onset joint degeneration and lifelong joint pain, is caused by mutations in cartilage oligomeric matrix protein (COMP). The mechanisms underlying the mutant-COMP pathology have been defined using the MT-COMP mouse model of PSACH that has the common D469del mutation. Mutant-COMP protein does not fold properly, and it is retained in the rough endoplasmic reticulum (rER) of chondrocytes rather than being exported to the extracellular matrix (ECM), driving ER stress that stimulates oxidative stress and inflammation, driving a self-perpetuating cycle. CHOP (ER stress signaling protein) and TNFα inflammation drive high levels of mTORC1 signaling, shutting down autophagy and blocking ER clearance, resulting in premature loss of chondrocytes that negatively impacts linear growth and causes early joint degeneration in MT-COMP mice and PSACH. Previously, we have shown that resveratrol treatment from birth to 20 weeks prevents joint degeneration and decreases the pathological processes in articular chondrocytes. Resveratrol's therapeutic mechanism of action in the mutant-COMP pathology was shown to act by primarily stimulating autophagy and reducing inflammation. Importantly, we demonstrated that MT-COMP mice experience pain consistent with PSACH joint pain. Here, we show, in the MT-COMP mouse, that resveratrol treatment must begin within 4 weeks to preserve joint health and reduce pain. Resveratrol treatment started at 6 or 8 weeks (to 20 weeks) was not effective in preventing joint degeneration. Collectively, our findings in MT-COMP mice show that there is a postnatal resveratrol treatment window wherein the inevitable mutant-COMP joint degeneration and pain can be prevented.

CurQ+, a Next-Generation Formulation of Curcumin, Ameliorates Growth Plate Chondrocyte Stress and Increases Limb Growth in a Mouse Model of Pseudoachondroplasia.

Mutations in cartilage oligomeric matrix protein (COMP) causes protein misfolding and accumulation in chondrocytes that compromises skeletal growth and joint health in pseudoachondroplasia (PSACH), a severe dwarfing condition. Using the MT-COMP mice, a murine model of PSACH, we showed that pathological autophagy blockage was key to the intracellular accumulation of mutant-COMP. Autophagy is blocked by elevated mTORC1 signaling, preventing ER clearance and ensuring chondrocyte death. We demonstrated that resveratrol reduces the growth plate pathology by relieving the autophagy blockage allowing the ER clearance of mutant-COMP, which partially rescues limb length. To expand potential PSACH treatment options, CurQ+, a uniquely absorbable formulation of curcumin, was tested in MT-COMP mice at doses of 82.3 (1X) and 164.6 mg/kg (2X). CurQ+ treatment of MT-COMP mice from 1 to 4 weeks postnatally decreased mutant COMP intracellular retention, inflammation, restoring both autophagy and chondrocyte proliferation. CurQ+ reduction of cellular stress in growth plate chondrocytes dramatically reduced chondrocyte death, normalized femur length at 2X 164.6 mg/kg and recovered 60% of lost limb growth at 1X 82.3 mg/kg. These results indicate that CurQ+ is a potential therapy for COMPopathy-associated lost limb growth, joint degeneration, and other conditions involving persistent inflammation, oxidative stress, and a block of autophagy.

Joint Degeneration in a Mouse Model of Pseudoachondroplasia: ER Stress, Inflammation, and Block of Autophagy.

Pseudoachondroplasia (PSACH), a short limb skeletal dysplasia associated with premature joint degeneration, is caused by misfolding mutations in cartilage oligomeric matrix protein (COMP). Here, we define mutant-COMP-induced stress mechanisms that occur in articular chondrocytes of MT-COMP mice, a murine model of PSACH. The accumulation of mutant-COMP in the ER occurred early in MT-COMP articular chondrocytes and stimulated inflammation (TNFα) at 4 weeks, and articular chondrocyte death increased at 8 weeks while ER stress through CHOP was elevated by 12 weeks. Importantly, blockage of autophagy (pS6), the major mechanism that clears the ER, sustained cellular stress in MT-COMP articular chondrocytes. Degeneration of MT-COMP articular cartilage was similar to that observed in PSACH and was associated with increased MMPs, a family of degradative enzymes. Moreover, chronic cellular stresses stimulated senescence. Senescence-associated secretory phenotype (SASP) may play a role in generating and propagating a pro-degradative environment in the MT-COMP murine joint. The loss of CHOP or resveratrol treatment from birth preserved joint health in MT-COMP mice. Taken together, these results indicate that ER stress/CHOP signaling and autophagy blockage are central to mutant-COMP joint degeneration, and MT-COMP mice joint health can be preserved by decreasing articular chondrocyte stress. Future joint sparing therapeutics for PSACH may include resveratrol.

Primary Osteoarthritis Early Joint Degeneration Induced by Endoplasmic Reticulum Stress Is Mitigated by Resveratrol.

Increasing numbers of people are living with osteoarthritis (OA) due to aging and obesity, creating an urgent need for effective treatment and preventions. Two top risk factors for OA, age and obesity, are associated with endoplasmic reticulum (ER) stress. The I-ERS mouse, an ER stress-driven model of primary OA, was developed to study the role of ER stress in primary OA susceptibility. The I-ERS mouse has the unique ability to induce ER stress in healthy adult articular chondrocytes and cartilage, driving joint degeneration that mimics early primary OA. In this study, ER stress-induced damage occurred gradually and stimulated joint degeneration with OA characteristics including increased matrix metalloproteinase activity, inflammation, senescence, chondrocyte death, decreased proteoglycans, autophagy block, and gait dysfunction. Consistent with human OA, intense exercise hastened and increased the level of ER stress-induced joint damage. Notably, loss of a critical ER stress response protein (CHOP) largely ameliorated ER stress-stimulated OA outcomes including preserving proteoglycan content, reducing inflammation, and relieving autophagy block. Resveratrol diminished ER stress-induced joint degeneration by decreasing CHOP, TNFα, IL-1ß, MMP-13, pS6, number of TUNEL-positive chondrocytes, and senescence marker p16 INK4a. The finding, that a dietary supplement can prevent ER stressed-induced joint degeneration in mice, provides a preclinical foundation to potentially develop a prevention strategy for those at high risk to develop OA.

Resveratrol Reduces COMPopathy in Mice Through Activation of Autophagy.

Misfolding mutations in cartilage oligomeric matrix protein (COMP) cause it to be retained within the endoplasmic reticulum (ER) of chondrocytes, stimulating a multitude of damaging cellular responses including ER stress, inflammation, and oxidative stress, which ultimately culminates in the death of growth plate chondrocytes and pseudoachondroplasia (PSACH). Previously, we demonstrated that an antioxidant, resveratrol, substantially reduces the intracellular accumulation of mutant-COMP, dampens cellular stress, and lowers the level of growth plate chondrocyte death. In addition, we showed that resveratrol reduces mammalian target of rapamycin complex 1 (mTORC1) signaling, suggesting a potential mechanism. In this work, we investigate the role of autophagy in treatment of COMPopathies. In cultured chondrocytes expressing wild-type COMP or mutant-COMP, resveratrol significantly increased the number of Microtubule-associated protein 1A/1B-light chain 3 (LC3) vesicles, directly demonstrating that resveratrol-stimulated autophagy is an important component of the resveratrol-driven mechanism responsible for the degradation of mutant-COMP. Moreover, pharmacological inhibitors of autophagy suppressed degradation of mutant-COMP in our established mouse model of PSACH. In contrast, blockage of the proteasome did not substantially alter resveratrol clearance of mutant-COMP from growth plate chondrocytes. Mechanistically, resveratrol increased SIRT1 and PP2A expression and reduced MID1 expression and activation of phosphorylated protein kinase B (pAKT) and mTORC1 signaling in growth plate chondrocytes, allowing clearance of mutant-COMP by autophagy. Importantly, we show that optimal reduction in growth plate pathology, including decreased mutant-COMP retention, decreased mTORC1 signaling, and restoration of chondrocyte proliferation was attained when treatment was initiated between birth to 1 week of age in MT-COMP mice, translating to birth to approximately 2 years of age in children with PSACH. These results clearly demonstrate that resveratrol stimulates clearance of mutant-COMP by an autophagy-centric mechanism. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Novel mTORC1 Mechanism Suggests Therapeutic Targets for COMPopathies.

Cartilage oligomeric matrix protein (COMP) is a large, multifunctional extracellular protein that, when mutated, is retained in the rough endoplasmic reticulum (ER). This retention elicits ER stress, inflammation, and oxidative stress, resulting in dysfunction and death of growth plate chondrocytes. While identifying the cellular pathologic mechanisms underlying the murine mutant (MT)-COMP model of pseudoachondroplasia, increased midline-1 (MID1) expression and mammalian target of rapamycin complex 1 (mTORC1) signaling was found. This novel role for MID1/mTORC1 signaling was investigated since treatments shown to repress the pathology also reduced Mid1/mTORC1. Although ER stress-inducing drugs or tumor necrosis factor α (TNFα) in rat chondrosarcoma cells increased Mid1, oxidative stress did not, establishing that ER stress- or TNFα-driven inflammation alone is sufficient to elevate MID1 expression. Since MID1 ubiquitinates protein phosphatase 2A (PP2A), a negative regulator of mTORC1, PP2A was evaluated in MT-COMP growth plate chondrocytes. PP2A was decreased, indicating de-repression of mTORC1 signaling. Rapamycin treatment in MT-COMP mice reduced mTORC1 signaling and intracellular retention of COMP, and increased proliferation, but did not change inflammatory markers IL-16 and eosinophil peroxidase. Lastly, mRNA from tuberous sclerosis-1/2-null mice brain tissue exhibiting ER stress had increased Mid1 expression, confirming the relationship between ER stress and MID1/mTORC1 signaling. These findings suggest a mechanistic link between ER stress and MID1/mTORC1 signaling that has implications extending to other conditions involving ER stress.

Mutant cartilage oligomeric matrix protein (COMP) compromises bone integrity, joint function and the balance between adipogenesis and osteogenesis.

Mutations in COMP (cartilage oligomeric matrix protein) cause severe long bone shortening in mice and humans. Previously, we showed that massive accumulation of misfolded COMP in the ER of growth plate chondrocytes in our MT-COMP mouse model of pseudoachondroplasia (PSACH) causes premature chondrocyte death and loss of linear growth. Premature chondrocyte death results from activation of oxidative stress and inflammation through the CHOP-ER pathway and is reduced by removing CHOP or by anti-inflammatory or antioxidant therapies. Although the mutant COMP chondrocyte pathologic mechanism is now recognized, the effect of mutant COMP on bone quality and joint health (laxity) is largely unknown. Applying multiple analytic approaches, we describe a novel mechanism by which the deleterious consequences of mutant COMP retention results in upregulation of miR-223 disturbing the adipogenesis - osteogenesis balance. This results in reduction in bone mineral density, bone quality, mechanical strength and subchondral bone thickness. These, in addition to abnormal patterns of ossification at the ends of the femoral bones likely contribute to precocious osteoarthritis (OA) of the hips and knees in the MT-COMP mouse and PSACH. Moreover, joint laxity is compromised by abnormally thin ligaments. Altogether, these novel findings align with the PSACH phenotype of delayed ossification and bone age, extreme joint laxity and joint erosion, and extend our understanding of the underlying processes that affect bone in PSACH. These results introduce a novel finding that miR-223 is involved in the ossification defect in MT-COMP mice making it a therapeutic target.

The astrocyte-expressed integrin αvß8 governs blood vessel sprouting in the developing retina.

The mouse retina is vascularized after birth when angiogenic blood vessels grow and sprout along a pre-formed latticework of astrocytes. How astrocyte-derived cues control patterns of blood vessel growth and sprouting, however, remains enigmatic. Here, we have used molecular genetic strategies in mice to demonstrate that αvß8 integrin expressed in astrocytes is essential for neovascularization of the developing retina. Selective ablation of αv or ß8 integrin gene expression in astrocytes leads to impaired blood vessel sprouting and intraretinal hemorrhage, particularly during formation of the secondary vascular plexus. These pathologies correlate, in part, with diminished αvß8 integrin-mediated activation of extracellular matrix-bound latent transforming growth factor ßs (TGFßs) and defective TGFß signaling in vascular endothelial cells, but not astrocytes. Collectively, our data demonstrate that αvß8 integrin is a component of a paracrine signaling axis that links astrocytes to blood vessels and is essential for proper regulation of retinal angiogenesis.

Glioblastoma angiogenesis and tumor cell invasiveness are differentially regulated by ß8 integrin.

Glioblastoma multiforme (GBM) is a highly invasive brain tumor that develops florid microvascular proliferation and hemorrhage. However, mechanisms that favor invasion versus angiogenesis in this setting remain largely uncharacterized. Here, we show that integrin ß8 is an essential regulator of both GBM-induced angiogenesis and tumor cell invasiveness. Highly angiogenic and poorly invasive tumors expressed low levels of ß8 integrin, whereas highly invasive tumors with limited neovascularization expressed high levels of ß8 integrin. Manipulating ß8 integrin protein levels altered the angiogenic and invasive growth properties of GBMs, in part, reflected by a diminished activation of latent TGFßs, which are extracellular matrix protein ligands for ß8 integrin. Taken together, these results establish a role for ß8 integrin in differential control of angiogenesis versus tumor cell invasion in GBM. Our findings suggest that inhibiting ß8 integrin or TGFß signaling may diminish tumor cell invasiveness during malignant progression and following antivascular therapies.

Analysis of αv integrin protein expression in human eyelid and periorbital squamous cell carcinomas.

BACKGROUND: Alpha v integrins are receptors for many extracellular matrix (ECM) protein ligands, including latent transforming growth factor betas (TGFßs). Various studies in mice have shown that ablation of genes encoding αv integrin or TGFß signaling pathway components leads to spontaneous squamous cell carcinomas (SCCs) in the conjunctiva and periocular skin. Here, we have analyzed patterns of αv integrin protein expression and TGFß signaling in human eyelid and periorbital SCC samples. METHODS: An anti-αv integrin antibody was used to immunostain 19 eyelid and periorbital SCC samples. Additionally, tissue lysates from resected normal eyelid and SCC samples were analyzed by immunoblotting for αv integrin protein. Tumor sections were also immunostained with an antibody directed against Smad2, an intracellular signaling protein that is phosphorylated by TGFß receptors. RESULTS: Alpha v integrin protein was highly expressed in the invasive and less-differentiated components of human SCCs. Lower levels of αv integrin protein were detected in more differentiated components of tumors, as well as in SCC in situ. Patterns of phosphorylated Smad2 immunoreactivity correlated with levels αv integrin expression. CONCLUSIONS: Alpha v integrin was expressed at robust levels in tumor cells representing less differentiated, more invasive components of SCC; by contrast, well-differentiated cells as well as SCC in situ expressed low levels of αv integrin protein.

Beta8 integrin regulates neurogenesis and neurovascular homeostasis in the adult brain.

Central nervous system (CNS) neurovascular units are multicellular complexes consisting of neural cells, blood vessels and a milieu of extracellular matrix (ECM) proteins. ECM-mediated adhesion and signaling events within neurovascular units probably contribute to proper CNS development and physiology; however, the molecular mechanisms that control these events remain largely undetermined. Previous studies from our group and others showed that ablation of the ECM receptor, alphavbeta8 integrin, in neural progenitor cells (NPCs) of the embryonic mouse brain results in severe developmental neurovascular pathologies and premature death. Here, we have investigated the functions for this integrin in the adult brain by studying mice harboring a homozygous-null beta8 gene mutation generated on an outbred background that permits survival for several months. We show that adult beta8-/- mice display widespread defects in neurovascular unit homeostasis, including increased numbers of intracerebral blood vessels with pronounced perivascular astrogliosis. Furthermore, in neurogenic regions of the adult brain, where NPCs cluster around blood vessels in neurovascular niches, beta8 integrin is essential for normal control of NPC proliferation and survival. Analysis of NPCs cultured ex vivo reveals that the growth and survival defects correlate, in part, with diminished integrin-mediated activation of latent transforming growth factor beta1 (TGFbeta1), which is an ECM protein ligand for alphavbeta8 integrin. Collectively, these data identify essential functions for beta8 integrin in regulating neurovascular unit physiology in the post-natal mouse brain.