Objectives: It has been reported that pain occurs after the onset of OA and is often associated with inflammatory synovial expression of tumor necrotic factor (TNFα), suggesting that TNFα is one of the main factors causing inflammation, pain and OA development in the joints (1). Inhibition of TNFα could be a potential approach to reduce inflammation in patients with OA. However, most anti-TNFα treatments in clinical trials with antibodies or inhibitors to reduce inflammation in OA have yielded conflicting results (2). It is essential to explore novel and more efficient approaches to modulate the expression of TNFα and inflammation in patients with painful OA. TNFα-induced protein 8-like 2 (TIPE2) was found to regulate the immune system’s homeostasis, and thus regulate inflammation. Zmpste24 deficient mouse (Z24-/-) is a reliable model of human Hutchinson-Gilford progeria. They are incapable of producing lamin A, an essential component of the nuclear envelope, and exhibit profound nuclear architecture abnormalities and multiple histopathological defects that phenocopy an accelerated aging process (3). Z24-/- mice are spontaneously and progressively developed OA around three months old. To investigate the role of TIPE2 on OA in this accelerated aging model, we have performed intra-articular injections of adeno-associated virus(AAV) carrying the TIPE2 gene in the knee of Z24-/- mice. Our results indicate that the overexpression of TIPE2 ameliorates osteoarthritis phenotype through the reduction of inflammation and senescent cells, suggesting that TIPE2 gene delivery may provide a novel anti-inflammatory therapy to alleviate the aging related OA in the clinic. Methods: Construction of the AAV-TIPE2 gene expression vector. Construction was performed as previously described (4). AAV production and titration. Viral production and purification of AAV2 were carried out as previously described (5). The concentration was determined using an AAV titration kit (Takara). The titer for AAV2-TIPE2 was 4.7 × 10⁁12 GCP/ml. Animals. Both male and female Z24-/- (B6.129SZmpste24tm1Sgy/Mmucd) mice at 12 weeks old were used for this study. The animal protocol was approved by the Institutional Animal Welfare Committee at the Colorado State University. TIPE2 treatment. AAV2-TIPE2 (20ul) was intra-articular injected into the right knee joint of Z24-/- mice and the same volume of PBS was injected into their left knee joint, which was used as control. At four weeks post-injection, mice were euthanized, and the knee joints were excised for decalcification, paraffin embedding, sectioning and histology analysis. Histology. H&E and Safranin O staining were performed following the manufacture protocol. The AC damage shown in the Safranin O staining images will be further evaluated using the Mankin scoring system(6). Immune staining. TNF-α and p16 immune staining was performed as the previous description(7). Immunofluorescence images were taken using a Nikon upright microscope. β-Gal staining. β-Gal staining was performed using the senescence staining kit (Cell Signaling). Statistical analysis. All values are expressed as the means ± SD. Statistical analyses were performed using Microsoft Excel software. Data were analyzed by the independent samples t-test compared between 2 groups at each time point using 2 tails. A value of P < 0.05 was considered statistically significant. Results: TIPE2 alleviates OA progression by regulating cell inflammatory responses. Since the inflammatory factor TNFα plays a key role in OA progression we first confirmed that TNFα is highly expressed in OA chondrocytes in the knee joint of Z24-/- mice at three months old (Fig.1). As we expected, TIPE2 treatment significantly decreased TNFα expression in chondrocytes of the OA knee (Fig. 1). TIPE2 treatment prevents cartilage degradation. We saw the difference in Safranin O–positive staining intensity in the AC (articular cartilage) of TIPE2 treated mice and PBS control mice. The glycosaminoglycan content (red) is degraded in the Z24-/- cartilage (PBS control), but cartilage degradation was reduced in Z24-/- mice treated with TIPE2 (Fig.2). TIPE2 treatment decreased β-Gal + senescent cells in the OA knee. OA as a whole joint disease, we observed the Z24 -/- mice chondrocytes exhibiting a variety of senescent-associated phenotypes. β-gal staining was observed in a subset of chondrocytes and subchondral bone close to the lesion sites of the OA. TIPE2 alleviates OA progression by targeting senescent cells. We observed that a significant decrease of TNFα positive cells (Fig. 1) and β-Gal positive cells (yellow arrow indicated in Fig.3, # p<0.05) by intra-articular injection of AAV TIPE2. This decrease is further confirmed by the determination of p16 expression in the Z24 -/- mice by TIPE2 treatment (Fig.3, *p<0.03). These findings suggest that TIPE2 can suppress OA progression via targeting inflammation factor TNFα in vivo. Conclusions: OA has become an emerging health challenge that occurs in older people. TNF-α is one of the main factors causing inflammation, pain and OA development in the joints. Here we first report that OA spontaneously occurred in aging joints of Z24-/- mice, and using a TNF-α antagonist, TIPE2, could attenuate OA development by decreasing inflammation through a reduction in the number of senescent cells. It has been reported that cellular senescence and the senescence-associated secretory phenotype (SASP) that drive and promote chronic inflammation in multiple age-related chronic diseases(8). Our results provide evidence that the downregulation of the inflammation factor TNF-α is capable to reduce cell senescence in this OA animal model. In conclusion, our research demonstrated a connection between TNF-α and senescence in aging caused OA. Our results indicate that TIPE2, as a TNF-α regulator, modulates inflammation can be used as a treatment for OA. [Figure: see text][Figure: see text][Figure: see text]
Objectives: Anterior cruciate ligament (ACL) reconstruction is the 6th most common orthopedic procedure performed in the United States (1,2). There is substantial evidence to suggest that muscle weakness significantly contributes to adverse outcomes after ACL injury/reconstruction (3). Despite efforts to improve rehabilitation methods, there are currently no effective strategies for restoring pre-injury muscle strength in ACL-injured limbs. Our team has identified that estrogen-related receptor gamma (ERRγ) is a crucial regulator of paracrine angiogenesis in the skeletal muscle (4). Selective over-expression of ERRγ in the skeletal muscle [ERRGO mice] activates a robust paracrine angiogenic gene program involving myofibrillar induction and secretion of a battery of angiogenic factors resulting in muscle vascularization (4). To determine if muscle ERRγ-driven angiogenesis can mitigate muscle atrophy after ACL injury, we performed ACL injury on the ERRGO mice, as well as age-matched wild-type (WT) littermate control mice. In this model, we found that ERRGO mice with muscle ERRγ overexpression significantly mitigated muscle atrophy compared to WT control mice 4 weeks after ACL injury. This finding strongly suggests that muscle-specific ERRγ activation may reduce muscle atrophy after ACL injury as a consequence of increased muscle angiogenesis. This preventive effect is potentially linked to developing a therapeutic approach to reverse these muscle changes after ACL surgery. Methods:Animals: 12 weeks old male and female ERRGO and WT mice obtained from Dr. Narkar’s laboratory were used for this study. The ACL injury was conducted as previously described (5). We performed ACL injury on the right leg, and the left leg was used as non-injured control. The mice were euthanized four weeks after injury. The muscle tissues were harvested, the gastrocnemius muscle (GM) mass was weighted, flash-frozen in liquid nitrogen-cooled 2-methylbutane, and cryo-sectioned. H&E staining was performed on 10 µm cryosections from GM according to the manufacturer’s instructions. Immunohistochemical staining: The muscle sections were fixed with 4% paraformaldehyde. A Mouse on Mouse kit (Vector) was used for anti-muscle RING-finger protein-1 (MuRF1, marker for muscle atrophy) staining according to the manufacturer’s protocol. Statistical analysis: All results are presented as mean ± standard deviation (SD). Means from ACL injured and non-injured of WT and ERRGO mice were compared using Student’s t-test. Differences were considered statistically significant when the P-value was < 0.05. Results: We performed the following experiments to determine if ERRγ overexpression in the muscle can prevent muscle weakness after ACL injury. The ACLs on the right leg of ERRGO and WT mice were excised. 4 weeks after injury, the mice were sacrificed, and muscle tissues were collected for histology analysis. First, we observed that muscles in the hindlimbs of WT mice were atropied, as expected, after ACL injury compared to the muscles in the non-injured hindlimb (Fig.1A). Strikingly, after ACL injury, the hindlimb muscles in ERRGO mice were resiliant to atrophy (Fig.1A). Quantitatively, we found that the gastrocnemius muscles weights were significantly reduced in WT mice after ACL injury compared to the GM weights from the non-injured leg. However, this ACL injury-induced reduction in gastrocnemius weight was not observed in ERRGO mice after ACL injury (Fig.1 B). The myofiber cross-sectional area (CSA) was measured based on the H&E staining on the GM muscle of ERRGO and WT mice to evaluate the muscle atrophy further. We found that the CSA of muscle fibers in WT mice was significantly smaller after ACL injury than in the non-injured control muscle (Fig. 2A, 2C, P<0.05). The average size of muscle fibers was not significantly decreased in the muscle of ERRGO mice after ACL injury compared to non-injured muscle. (Fig. 2A, 2C, P>0.05). Since MuRF1 is a biomarker of myofiber atrophy (6), we evaluated the MuRF1 expression in muscle sections by immunostaining. The result showed an increase in MuRF1 expression in the WT muscle compared to ERRGO muscle after ACL injury (Fig. 2B). Together, those results demonstrated that muscle-specific ERRγ activation mitigates muscle atrophy after ACL injury. Conclusions: Skeletal muscle is adversely affected by the ACL injury, and post-reconstruction recovery is limited by muscle weakness. It has been reported that ERRγ expression in the skeletal muscle directly correlates with vascular density, and ERRγ is highly expressed in well-vascularized muscle beds (4). Based on our preliminary data, we observed that the ERRGO mice with muscle-specific ERRγ activation have the capacity to mitigate the muscle atrophy after ACL injury. As we know, exercise induces muscle angiogenesis, and regular physical activity has been considered a therapeutic modality for preventing aging-related muscle wasting. Although exercise is the primary method for alleviating muscle weakness, many patients cannot achieve the exercise intensity that is necessary to prevent or reverse muscle atrophy. Drugs targeting ERRγ will likely be safe as ERRγ belongs to the nuclear receptor superfamily, which are excellent ‘druggable’ targets with unique ligand-binding pockets that facilitate selective and specific drug design. Future studies will investigate the beneficial effects of ERRγ overexpression in ERRGO mice on muscle atrophy after ACL injury at different time points and determine if muscle-specific activation of ERRγ can mitigate age-related muscle progenitor cells dysfunction and offset the infiltration and activation of FAPs and senescent cells after ACL injury
Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we utilized an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 can be used to successfully deliver Pdx1 and MafA into alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells was also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.
Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we utilized an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 can be used to successfully deliver Pdx1 and MafA into alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells was also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.
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