Silencing VEGFR-2 Hampers Odontoblastic Differentiation of Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.

Down-regulated Treg cells in exacerbated periodontal disease during pregnancy.

Pregnancy is a special period marked with complicated changes in various immune responses. Although pregnant women are prone to developing gingival inflammation, its immunological mechanism remains to be clarified. In a modified ligature-induced periodontal disease murine model, pregnant mice developed more severe alveolar bone loss. Using this model, we investigated the Treg responses during exacerbated periodontal disease in pregnant mice. We tested Treg-associated molecules in gingival tissues by quantitative real-time PCR and found decreased gingival expression of Foxp3, TGFß, CTLA-4, and CD28 in pregnant mice after periodontal disease induction. We further confirmed that lower number of Treg cells were present in the cervical lymph nodes of pregnant periodontitis mice. Treg cells from the cervical lymph nodes of ligated pregnant mice and non-pregnant mice were tested for their suppressive function in vitro. We manifested that Treg suppressive function was also down-regulated in the pregnant mice. Additionally, we demonstrated that more inflammatory Th17 cells were present in the cervical lymph nodes of ligated pregnant mice. Therefore, impaired Treg development and function, together with upregulated Th17 response, may contribute to the exacerbated periodontal disease during pregnancy.

Porphyromonas gingivalis induces exacerbated periodontal disease during pregnancy.

Although pregnant women are prone to gingival inflammation, its mechanism remains unclear. Animal models are ideal for investigating immunological mechanisms in the periodontal disease. A murine model for ligature-induced periodontal disease has been modified and utilized to determine the susceptibility to periodontal inflammation and tissue damage in pregnant mice. Expression of different inflammatory mediators in the gingivae was determined by quantitative real-time PCR (qPCR). Inflammatory bone loss was determined by measuring the distance from the cementoenamel junction to the alveolar bone crest (CEJ-ABC). Oral bacterial number was determined by the CFU (Colony Forming Units) count from anaerobic culture of oral swabs. In our experiments, ligation itself did not cause higher gingival inflammation and bone loss in pregnant mice than non-pregnant mice, while ligation combined with P. gingivalis infection led to increased gingival inflammation and periodontal bone loss, accompanied by lower gingival expression of anti-inflammatory cytokines in pregnant mice. Our results indicated that P. gingivalis infection was important in inducing more severe periodontal diseases during pregnancy, which might be attributed to the down-regulated anti-inflammatory mechanisms, but not be associated with higher oral bacterial burden.

MicroRNA-21 down-regulates inflammation and inhibits periodontitis.

Periodontitis is one of the most prevalent inflammatory diseases, characterized by gingival inflammation and alveolar bone loss. MicroRNAs (MiRNAs) are important regulators of inflammation and involved in periodontitis pathogenesis. In this work, we studied the roles of microRNA-21 (miR-21) in periodontitis. MiR-21 is up-regulated in both periodontitis patients and the mice that induced with periodontitis. We tested the roles of miR-21 in the macrophages challenged by periodontitis pathogen Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). MiR-21 expression is up-regulated in P. gingivalis LPS-stimulated macrophages. MiR-21 mimic inhibits the pro-inflammatory cytokine production by macrophages, while miR-21 deficiency elevates the production of pro-inflammatory cytokines. Moreover, absence of miR-21 promotes activation of nuclear factor-κB (NF-κB) in P. gingivalis LPS- stimulated cells. In a murine periodontitis model, ligation induced exacerbated gingival inflammation and alveolar bone loss in miR-21 deficient mice than their wild-type littermates. These results demonstrated the anti-inflammatory function of miR-21 in vitro and in vivo, indicating miR-21 could be an interventional target for the control of periodontitis.

Harnessing Sphingosine-1-Phosphate Signaling and Nanotopographical Cues To Regulate Skeletal Muscle Maturation and Vascularization.

Despite possessing substantial regenerative capacity, skeletal muscle can suffer from loss of function due to catastrophic traumatic injury or degenerative disease. In such cases, engineered tissue grafts hold the potential to restore function and improve patient quality of life. Requirements for successful integration of engineered tissue grafts with the host musculature include cell alignment that mimics host tissue architecture and directional functionality, as well as vascularization to ensure tissue survival. Here, we have developed biomimetic nanopatterned poly(lactic-co-glycolic acid) substrates conjugated with sphingosine-1-phosphate (S1P), a potent angiogenic and myogenic factor, to enhance myoblast and endothelial maturation. Primary muscle cells cultured on these functionalized S1P nanopatterned substrates developed a highly aligned and elongated morphology and exhibited higher expression levels of myosin heavy chain, in addition to genes characteristic of mature skeletal muscle. We also found that S1P enhanced angiogenic potential in these cultures, as evidenced by elevated expression of endothelial-related genes. Computational analyses of live-cell videos showed a significantly improved functionality of tissues cultured on S1P-functionalized nanopatterns as indicated by greater myotube contraction displacements and velocities. In summary, our study demonstrates that biomimetic nanotopography and S1P can be combined to synergistically regulate the maturation and vascularization of engineered skeletal muscles.

PDGFRα signalling promotes fibrogenic responses in collagen-producing cells in Duchenne muscular dystrophy.

Fibrosis is a characteristic of Duchenne muscular dystrophy (DMD), yet the cellular and molecular mechanisms responsible for DMD fibrosis are poorly understood. Utilizing the Collagen1a1-GFP transgene to identify cells producing Collagen-I matrix in wild-type mice exposed to toxic injury or those mutated at the dystrophin gene locus (mdx) as a model of DMD, we studied mechanisms of skeletal muscle injury/repair and fibrosis. PDGFRα is restricted to Sca1+, CD45- mesenchymal progenitors. Fate-mapping experiments using inducible CreER/LoxP somatic recombination indicate that these progenitors expand in injury or DMD to become PDGFRα+, Col1a1-GFP+ matrix-forming fibroblasts, whereas muscle fibres do not become fibroblasts but are an important source of the PDGFRα ligand, PDGF-AA. While in toxin injury/repair of muscle PDGFRα, signalling is transiently up-regulated during the regenerative phase in the DMD model and in human DMD it is chronically overactivated. Conditional expression of the constitutively active PDGFRα D842V mutation in Collagen-I+ fibroblasts, during injury/repair, hindered the repair phase and instead promoted fibrosis. In DMD, treatment of mdx mice with crenolanib, a highly selective PDGFRα/ß tyrosine kinase inhibitor, reduced fibrosis, improved muscle strength, and was associated with decreased activity of Src, a downstream effector of PDGFRα signalling. These observations are consistent with a model in which PDGFRα activation of mesenchymal progenitors normally regulates repair of the injured muscle, but in DMD persistent and excessive activation of this pathway directly drives fibrosis and hinders repair. The PDGFRα pathway is a potential new target for treatment of progressive DMD. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Chlamydia pneumoniae infection of lungs and macrophages indirectly stimulates the phenotypic conversion of smooth muscle cells and mesenchymal stem cells: potential roles in vascular calcification and fibrosis.

Two hallmarks of advanced atherosclerosis are calcification and fibrosis. We hypothesized that Chlamydia pneumoniae infection may contribute to atherosclerosis by inducing the conversion of vascular smooth muscle cells to calcifying cells or by converting mesenchymal stem cells to osteochondrocytic or fibroblastic phenotypes. In this study, direct infection of bovine aortic smooth muscle cells (BSMCs) did not induce the expression of alkaline phosphatase or the deposition of extracellular calcium phosphate. However, conditioned media from C. pneumoniae-infected macrophages accelerated conversion of BSMCs to a calcifying phenotype. Treatment of the conditioned media with an anti-TNF-alpha blocking antibody abrogated this stimulatory effect. Treatment of perivascular Sca-1+, CD31-, CD45- cells from apoE-/- mouse aortas with the conditioned media from infected macrophages induced the Sca-1+ cells to produce collagen II, an additional marker of an osteochondrocytic phenotype. Treatment of mouse coronary perivascular Sca-1+, CD31-, CD45- cells with the supernatant from homogenates of C. pneumoniae-infected mouse lungs as compared to noninfected lungs induced expression of the Collagen 1α1 gene and deposition of collagen. Therefore, an increase in plasma cytokines or other factors in response to respiratory infection with C. pneumoniae or infection of macrophages within the blood vessel could contribute to both calcification and fibrosis of advanced atherosclerotic lesions.

Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease. Studies in Drosophila showed that genetic increase of the levels of the bioactive sphingolipid sphingosine-1-phosphate (S1P) or delivery of 2-acetyl-5-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, suppresses dystrophic muscle degeneration. In the dystrophic mouse (mdx), upregulation of S1P by THI increases regeneration and muscle force. S1P can act as a ligand for S1P receptors and as a histone deacetylase (HDAC) inhibitor. Because Drosophila has no identified S1P receptors and DMD correlates with increased HDAC2 levels, we tested whether S1P action in muscle involves HDAC inhibition. Here we show that beneficial effects of THI treatment in mdx mice correlate with significantly increased nuclear S1P, decreased HDAC activity and increased acetylation of specific histone residues. Importantly, the HDAC2 target microRNA genes miR-29 and miR-1 are significantly upregulated, correlating with the downregulation of the miR-29 target Col1a1 in the diaphragm of THI-treated mdx mice. Further gene expression analysis revealed a significant THI-dependent decrease in inflammatory genes and increase in metabolic genes. Accordingly, S1P levels and functional mitochondrial activity are increased after THI treatment of differentiating C2C12 cells. S1P increases the capacity of the muscle cell to use fatty acids as an energy source, suggesting that THI treatment could be beneficial for the maintenance of energy metabolism in mdx muscles.

Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice.

BACKGROUND: Presently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice. METHODS: We treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles. RESULTS: Short-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function. CONCLUSIONS: These data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD.

Coronary adventitial cells are linked to perivascular cardiac fibrosis via TGFß1 signaling in the mdx mouse model of Duchenne muscular dystrophy.

In Duchenne muscular dystrophy (DMD), progressive accumulation of cardiac fibrosis promotes heart failure. While the cellular origins of fibrosis in DMD hearts remain enigmatic, fibrotic tissue conspicuously forms near the coronary adventitia. Therefore, we sought to characterize the role of coronary adventitial cells in the formation of perivascular fibrosis. Utilizing the mdx model of DMD, we have identified a population of Sca1+, PDGFRα+, CD31-, and CD45- coronary adventitial cells responsible for perivascular fibrosis. Histopathology of dystrophic hearts revealed that Sca1+ cells extend from the adventitia and occupy regions of perivascular fibrosis. The number of Sca1+ adventitial cells increased two-fold in fibrotic mdx hearts vs. age matched wild-type hearts. Moreover, relative to Sca1-, PDGFRα+, CD31-, and CD45- cells and endothelial cells, Sca1+ adventitial cells FACS-sorted from mdx hearts expressed the highest level of Collagen1α1 and 3α1, Connective tissue growth factor, and Tgfßr1 transcripts. Surprisingly, mdx endothelial cells expressed the greatest level of the Tgfß1 ligand. Utilizing Collagen1α1-GFP reporter mice, we confirmed that the majority of Sca1+ adventitial cells expressed type I collagen, an abundant component of cardiac fibrosis, in both wt (71%±4.1) and mdx (77%±3.5) hearts. In contrast, GFP+ interstitial fibroblasts were PDGFRα+ but negative for Sca1. Treatment of cultured Collagen1α1-GFP+ adventitial cells with TGFß1 resulted in increased collagen synthesis, whereas pharmacological inhibition of TGFßR1 signaling reduced the fibrotic response. Therefore, perivascular cardiac fibrosis by coronary adventitial cells may be mediated by TGFß1 signaling. Our results implicate coronary endothelial cells in mediating cardiac fibrosis via transmural TGFß signaling, and suggest that the coronary adventitia is a promising target for developing novel anti-fibrotic therapies.

An in vitro culture system for long-term expansion of epithelial and mesenchymal salivary gland cells: role of TGF-ß1 in salivary gland epithelial and mesenchymal differentiation.

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-ß1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-ß1 on salivary gland cell differentiation. TGF-ß1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-ßR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-ß signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-ß1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors.

Bone marrow-derived cells do not engraft into skeletal muscle microvasculature but promote angiogenesis after acute injury.

The skeletal muscle is supported by a vast network of microvessels with the capacity to regenerate in response to injury. However, the dynamics of microvascular repair and the origin of reconstituted endothelial cells in the skeletal muscle are poorly understood. A growing body of literature exists to indicate bone marrow (BM)-derived cells engraft into regenerating vascular endothelium and muscle macrovasculature. Therefore, we investigated the extent of BM contribution to skeletal muscle microvasculature after acute injury. Because reporters and markers commonly used to trace donor BM cells are not endothelial specific but are also expressed by leukocytes, we generated novel BM chimeras utilizing Tie2-green fluorescent protein BM cells transplanted into CD31 and Caveolin-1 knockout recipients. In turn, we surveyed BM vascular contribution, not just by the presence of green fluorescent protein, but also CD31 and Caveolin-1, respectively. After stable BM reconstitution, chimera limb muscles were cardiotoxin (CTX) injured and examined 21 days post-injury for the presence of green fluorescent protein, CD31, and Caveolin-1. Acute muscle injury by CTX is characterized by initial microvasculature death followed by rapid endothelial regeneration within 14 days post-damage. Histological analysis of injured and uninjured contralateral limb muscles revealed a complete absence of BM engraftment in the muscle vasculature of wild-type and CD31/Caveolin-1 knockout chimeras. In contrast, F4/80(+) cells isolated from CTX-injured muscle, expressed endothelial-related markers and promoted angiogenesis in vitro. Therefore, despite the absence of BM engraftment to regenerated skeletal muscle microvasculature, macrophages recruited after injury promote angiogenesis and, in turn, vascular regeneration.