Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat.

Brown fat is specialized for energy expenditure and has therefore been proposed to function as a defense against obesity. Despite recent advances in delineating the transcriptional regulation of brown adipocyte differentiation, cellular lineage specification and developmental cues specifying brown-fat cell fate remain poorly understood. In this study, we identify and isolate a subpopulation of adipogenic progenitors (Sca-1(+)/CD45(-)/Mac1(-); referred to as Sca-1(+) progenitor cells, ScaPCs) residing in murine brown fat, white fat, and skeletal muscle. ScaPCs derived from different tissues possess unique molecular expression signatures and adipogenic capacities. Importantly, although the ScaPCs from interscapular brown adipose tissue (BAT) are constitutively committed brown-fat progenitors, Sca-1(+) cells from skeletal muscle and subcutaneous white fat are highly inducible to differentiate into brown-like adipocytes upon stimulation with bone morphogenetic protein 7 (BMP7). Consistent with these findings, human preadipocytes isolated from subcutaneous white fat also exhibit the greatest inducible capacity to become brown adipocytes compared with cells isolated from mesenteric or omental white fat. When muscle-resident ScaPCs are re-engrafted into skeletal muscle of syngeneic mice, BMP7-treated ScaPCs efficiently develop into adipose tissue with brown fat-specific characteristics. Importantly, ScaPCs from obesity-resistant mice exhibit markedly higher thermogenic capacity compared with cells isolated from obesity-prone mice. These data establish the molecular characteristics of tissue-resident adipose progenitors and demonstrate a dynamic interplay between these progenitors and inductive signals that act in concert to specify brown adipocyte development.

INZ-701 Prevents Ectopic Tissue Calcification and Restores Bone Architecture and Growth in ENPP1-Deficient Mice.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the major enzyme that cleaves extracellular adenosine triphosphate (ATP) to generate pyrophosphate (PPi), an inorganic metabolite with potent anticalcification activity. Loss-of-function mutations cause hypopyrophosphatemia and lead to a state of ENPP1 deficiency, which has an acute infantile phase known as generalized arterial calcification of infancy (GACI) and a pediatric to adult phase known as autosomal-recessive hypophosphatemic rickets type 2 (ARHR2). ENPP1 deficiency manifests as ectopic calcification of multiple tissues, neointimal proliferation, premature mortality, impaired growth, and bone deformities. INZ-701, a human ENPP1-Fc protein, is in clinical development as an enzyme replacement therapy for the treatment of ENPP1 deficiency. The pharmacokinetic and pharmacodynamic profile and therapeutic effect of INZ-701 were investigated in Enpp1asj/asj mice, a murine model of ENPP1 deficiency. Enpp1asj/asj mice have undetectable plasma PPi, lower plasma phosphate, and higher FGF23 levels compared with wild-type (WT) mice. Enpp1asj/asj mice on the acceleration diet, containing high phosphate and low magnesium, quickly develop clinical signs, including dehydration, rough hair coat, pinned ears, stiffed legs, and hunched back. Enpp1asj/asj mice treated with vehicle had aforementioned clinical signs plus severe ectopic calcification in multiple tissues and bone defects, characteristics of the clinical phenotype observed in GACI and ARHR2 patients. Our results showed a durable PPi response for more than 3 days after a single dose of INZ-701. Treatment of ENPP1-deficient mice every other day with INZ-701 for 8 weeks restored circulating levels of PPi, prevented pathological calcification in all the tested organs, restored growth parameters, corrected bone defects, improved clinical signs, and decreased mortality in Enpp1asj/asj mice, demonstrating the potential of INZ-701 to treat ENPP1 deficiency. © 2021 American Society for Bone and Mineral Research (ASBMR).

Central role of the MEK/ERK MAP kinase pathway in a mouse model of rheumatoid arthritis: potential proinflammatory mechanisms.

OBJECTIVE: To evaluate the role of the MEK/ERK MAP kinase pathway in murine collagen-induced arthritis (CIA) using the selective MEK inhibitor PD184352. We examined the effects of the inhibitor in cytokine-stimulated synovial fibroblasts and in cytokine-induced arthritis in rabbits to investigate its antiinflammatory mechanisms. METHODS: Murine CIA was used to assess the effects of the selective MEK inhibitor on paw edema, clinical scores, weight loss, histopathologic features, and joint levels of p-ERK. Western blotting and immunohistochemistry techniques were used to assess p-ERK in human and rabbit synovial fibroblasts and synovial tissue from rheumatoid arthritis (RA) patients. Interleukin-1alpha (IL-1alpha)-stimulated stromelysin production in rabbit synovial fibroblasts was assessed by enzyme-linked immunosorbent assay. A rabbit IL-1alpha-induced arthritis model was used to assess the effects of the inhibitor on IL-1alpha-induced MEK activity, stromelysin production, and cartilage degradation. RESULTS: In the CIA model, PD184352 inhibited paw edema and clinical arthritis scores in a dose-dependent manner. Disease-induced weight loss and histopathologic changes were also significantly improved by treatment. Inhibition of disease-induced p-ERK levels in the joints was seen with the inhibitor. Levels of p-ERK in the synovium were higher in RA patients than in normal individuals. PD184352 reduced IL-1alpha-induced p-ERK levels in human RA synovial fibroblasts. The production of p-ERK and stromelysin was also inhibited in IL-1alpha-stimulated rabbit synovial fibroblasts. We observed IL-1alpha-induced p-ERK in the synovial lining, subsynovial vasculature, and articular chondrocytes. IL-1alpha-induced stromelysin production and proteoglycan loss from the articular cartilage were reduced by PD184352. CONCLUSION: These data demonstrate the inhibition of murine CIA by PD184352, support the hypothesis that antiinflammatory activity contributes to the mechanism of action of the inhibitor, and suggest that a selective inhibitor may effectively treat RA and other inflammatory disorders.

Oral treatment with PD-0200347, an alpha2delta ligand, reduces the development of experimental osteoarthritis by inhibiting metalloproteinases and inducible nitric oxide synthase gene expression and synthesis in cartilage chondrocytes.

OBJECTIVE: To examine the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage-activated Ca(2+) channels and a compound chemically related to pregabalin and gabapentin, on the development of cartilage structural changes in an experimental dog model of osteoarthritis (OA). The effects of PD-0200347 on the major pathways involved in OA cartilage degradation, including matrix metalloproteinases (MMPs) and the inducible form of nitric oxide synthase (iNOS), were also studied. METHODS: OA was surgically induced in dogs by sectioning the anterior cruciate ligament. OA dogs were randomly distributed into 3 groups and treated orally with either 1) placebo, 2) 15 mg/kg/day of PD-0200347, or 3) 90 mg/kg/day of PD-0200347. Dogs were killed 12 weeks after surgery. The severity of the lesions was scored macroscopically and histologically. Cartilage specimens from the femoral condyles and tibial plateaus were processed for RNA extraction and quantitative reverse transcription-polymerase chain reaction (RT-PCR) or immunohistochemistry. Specific probes and antibodies were used to study the messenger RNA and protein levels of iNOS, MMP-1, MMP-3, and MMP-13. RESULTS: No clinical signs of drug toxicity were noted in the treated animals. Treatment with PD-0200347 at both dosages tested (15 and 90 mg/kg/day) reduced the development of cartilage lesions. There was a reduction in the score of lesions, with a statistically significant (P = 0.01) difference when the highest dosage of the drug was administered. The reduction in the score was mainly related to a decrease in the surface size of the lesions. Quantitative RT-PCR showed that PD-0200347 significantly reduced the expression of MMP-13, a key mediator in OA. Immunohistochemical analyses showed that treatment with PD-0200347 significantly reduced the synthesis of all key OA mediators studied. CONCLUSION: This study demonstrated the efficacy of PD-0200347 in reducing the progression of cartilage structural changes in a dog model of OA. It also showed that this effect is linked to the inhibition of the major pathophysiologic mediators responsible for cartilage degradation.

In vivo selective inhibition of mitogen-activated protein kinase kinase 1/2 in rabbit experimental osteoarthritis is associated with a reduction in the development of structural changes.

OBJECTIVE: The primary aim of this study was to investigate, using an experimental rabbit model of osteoarthritis (OA), the effect of a selective mitogen-activated protein kinase kinase 1/2 (MEK-1/2) inhibitor, PD 198306, on the development of structural changes. Additional aims were to assess the effects of the inhibitor on levels of phosphorylated extracellular signal-regulated kinase 1/2 (phospho-ERK-1/2) and matrix metalloproteinase 1 (MMP-1; collagenase 1) in OA chondrocytes. METHODS: After surgical sectioning of the anterior cruciate ligament of the right knee joint, rabbits with OA were separated into 3 experimental groups: oral treatment with placebo or with PD 198306 at a therapeutic concentration of 10 mg/kg/day or 30 mg/kg/day. Each treatment started immediately after surgery. The animals were killed 8 weeks after surgery. Macroscopic and histologic studies were performed on the cartilage and synovial membrane. The levels of phospho-ERK-1/2 and MMP-1 in OA cartilage chondrocytes were evaluated by immunohistochemistry. Normal, untreated rabbits were used as controls. RESULTS: OA rabbits treated with the highest dosage of MEK-1/2 inhibitor showed decreases in the surface area (size) of cartilage macroscopic lesions (P < 0.002) and in osteophyte width on the lateral condyles (P = 0.05). Histologically, the severity of synovial inflammation (villous hyperplasia) was also reduced (P < 0.02). In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in the superficial layer stained positive for phospho-ERK-1/2 and MMP-1 compared with normal controls. Rabbits treated with the highest dosage of PD 198306 demonstrated a significant and dose-dependent reduction in the level of phospho-ERK-1/2 and a lower level of MMP-1. CONCLUSION: This study demonstrates that, in vivo, PD 198306, a selective inhibitor of MEK-1/2, can partially decrease the development of some of the structural changes in experimental OA. This effect was associated with a reduction in the level of phospho-ERK-1/2 in OA chondrocytes, which probably explains the action of the drug.