IL4 receptor targeting enables nab-paclitaxel to enhance reprogramming of M2-type macrophages into M1-like phenotype via ROS-HMGB1-TLR4 axis and inhibition of tumor growth and metastasis.

Rationale: Nab-paclitaxel (Abx) is widely employed in malignant tumor therapy. In tumor cells and pro-tumoral M2-type macrophages, the IL4 receptor (IL4R) is upregulated. This study aimed to elucidate the selective delivery of Abx to M2-type macrophages by targeting IL4R and reprogramming them into an anti-tumoral M1-type. Methods: Abx was conjugated with the IL4R-binding IL4RPep-1 peptide using click chemistry (IL4R-Abx). Cellular internalization, macrophage reprogramming and signal pathways, and tumor growth and metastasis by IL4R-Abx were examined. Results: IL4R-Abx was internalized into M2 macrophages more efficiently compared to the unmodified Abx and control peptide-conjugated Abx (Ctrl-Abx), which was primarily inhibited using an anti-IL4R antibody and a receptor-mediated endocytosis inhibitor compared with a macropinocytosis inhibitor. IL4R-Abx reprogrammed the M2-type macrophages into M1-like phenotype and increased reactive oxygen species (ROS) levels and extracellular release of high mobility group box 1 (HMGB1) in M2 macrophages at higher levels than Abx and Ctrl-Abx. The conditioned medium of IL4R-Abx-treated M2 macrophages skewed M2 macrophages into the M1-like phenotype, in which an anti-HMGB1 antibody and a toll-like receptor 4 (TLR4) inhibitor induced a blockade. IL4R-Abx accumulated at tumors, heightened immune-stimulatory cells while reducing immune-suppressing cells, and hampered tumor growth and metastasis in mice more efficiently than Abx and Ctrl-Abx. Conclusions: These results indicate that IL4R-targeting allows enhancement of M2-macrophage shaping into M1-like phenotype by Abx through the ROS-HMGB1-TLR4 axis, improvement of antitumor immunity, and thereby inhibition of tumor growth and metastasis, presenting a new approach to cancer immunotherapy.

Damage associated molecular patterns in necrotic femoral head inhibit osteogenesis and promote fibrogenesis of mesenchymal stem cells.

In Legg-Calvé-Perthes disease (LCPD), a loss of blood supply to the juvenile femoral head leads to extensive cell death and release of damage-associated molecular patterns (DAMPs). Over time chronic inflammatory repair process is observed with impaired bone regeneration. Increased fibrous tissue and adipose tissue are seen in the marrow space with decreased osteogenesis in a piglet model of LCPD, suggesting inhibition of osteoblastic differentiation and stimulation of fibroblastic and adipogenic differentiation of mesenchymal stem cell (MSC) during the healing process. Little is known about the DAMPs present in the necrotic femoral head and their effects on MSC differentiation. The purpose of this study was to characterize the DAMPs present in the femoral head following ischemic osteonecrosis and to determine their effects on MSC differentiation. Necrotic femoral heads were flushed with saline at 48 h, 2 weeks and 4 weeks following the induction of ischemic osteonecrosis in piglets to obtain necrotic bone fluid (NBF). Western blot analysis of the NBF revealed the presence of prototypic DAMP, high mobility group box 1 (HMGB1), and other previously described DAMPs: biglycan, 4-hydroxynonenal (4-HNE), and receptor activator of NF-κB ligand (RANKL). ELISA of the NBF revealed increasing levels of inflammatory cytokines IL1ß, IL6 and TNFα with the temporal progression of osteonecrosis. To determine the effects of NBF on MSC differentiation, we cultured primary porcine MSCs with NBF obtained by in vivo necrotic bone flushing method. NBF inhibited osteoblastic differentiation of MSCs with significantly decreased OSX expression (p = 0.008) and Von Kossa/Alizarin Red staining for mineralization. NBF also significantly increased the expression of proliferation markers Ki67 (p = 0.03) and PCNA (p < 0.0001), and fibrogenic markers Vimentin (p = 0.02) and Fibronectin (p = 0.04). Additionally, NBF treated MSC cells showed significantly elevated RANKL/OPG secretion ratio (p = 0.003) and increased expression of inflammatory cytokines IL1ß (p = 0.006) and IL6 (p < 0.0001). To specifically assess the role of DAMPs in promoting the fibrogenesis, we treated porcine fibroblasts with artificial NBF produced by bone freeze-thaw method. We found increased fibroblastic cell proliferation in an NBF dose-dependent manner. Lastly, we studied the effect of HMGB1, a prototypic DAMP, and found that HMGB1 partially contributes to MSC proliferation and fibrogenesis. In summary, our findings show that DAMPs and the inflammatory cytokines present in the necrotic femoral head inhibit osteogenesis and promote fibrogenesis of MSCs, potentially contributing to impaired bone regeneration following ischemic osteonecrosis as observed in LCPD.

Minimally Invasive Necrotic Bone Washing Improves Bone Healing After Femoral Head Ischemic Osteonecrosis: An Experimental Investigation in Immature Pigs.

BACKGROUND: Ischemic osteonecrosis of the femoral head produces necrotic cell debris and inflammatory molecules in the marrow space, which elicit a chronic inflammatory repair response. The purpose of this study was to determine the effects of flushing out the necrotic cell debris and inflammatory proteins on bone repair in a piglet model of ischemic osteonecrosis. METHODS: Osteonecrosis of the femoral head of the right hindlimb was induced in 12 piglets by tying a ligature tightly around the femoral neck. One week after the surgery, 6 animals were treated with a percutaneous 3-needle bone washing procedure and non-weight-bearing (NWB) of the right hindlimb (wash group). The total saline solution wash volume was 450 mL per femoral head. Serial wash solutions were collected and analyzed. The remaining 6 animals were treated with NWB only (NWB group). At 8 weeks after the surgery, the femoral heads were assessed using radiography, micro-computed tomography (micro-CT), and histological analysis. In addition, we compared the results for these piglets with our published results for 6 piglets treated with multiple epiphyseal drilling (MED) plus NWB without bone washing (MED group). RESULTS: Necrotic cells and inflammatory proteins were present in the bone wash solution collected 1 week after ischemia induction. The protein and triglyceride concentrations decreased significantly with subsequent washing (p < 0.005). At 8 weeks after ischemia induction, the wash group had a significantly higher bone volume than the MED or NWB group (p < 0.0001). Histological bone-formation measures were also significantly increased in the wash group compared with the MED group (p = 0.002) or NWB group (p < 0.0001) while macrophage numbers were significantly decreased in the wash group. CONCLUSIONS: The percutaneous 3-needle procedure flushed out cell debris and inflammatory proteins from the necrotic femoral heads, decreased osteoclasts and macrophages, and increased bone formation following induction of ischemic osteonecrosis. CLINICAL RELEVANCE: We believe that this is the first study to investigate the concept of washing out the necrotic femoral head to improve bone healing. The minimally invasive procedure may be useful to improve the necrotic bone environment and bone repair following ischemic osteonecrosis.

A Flowable Placental Formulation Prevents Bleomycin-Induced Dermal Fibrosis in Aged Mice.

Fibrosis, the thickening and scarring of injured connective tissue, leads to a loss of organ function. Multiple cell types, including T-cells, macrophages, fibrocytes, and fibroblasts/myofibroblasts contribute to scar formation via secretion of inflammatory factors. This event results in an increase in oxidative stress and deposition of excessive extracellular matrix (ECM), characteristic of fibrosis. Further, aging is known to predispose connective tissue to fibrosis due to reduced tissue regeneration. In this study, we investigated the anti-fibrotic activity of a flowable placental formulation (FPF) using a bleomycin-induced dermal fibrosis model in aged mice. FPF consisted of placental amnion/chorion- and umbilical tissue-derived ECM and cells. The mice were injected with either FPF or PBS, followed by multiple doses of bleomycin. Histological assessment of FPF-treated skin samples revealed reduced dermal fibrosis, inflammation, and TGF-ß signaling compared to the control group. Quantitative RT-PCR and Next Generation Sequencing analysis of miRNAs further confirmed anti-fibrotic changes in the FPF-treated group at both the gene and transcriptional levels. The observed modulation in miRNAs was associated with inflammation, TGF-ß signaling, fibroblast proliferation, epithelial-mesenchymal transition and ECM deposition. These results demonstrate the potential of FPF in preventing fibrosis and may be of therapeutic benefit for those at higher risk of fibrosis due to wounds, aging, exposure to radiation and genetic predisposition.

Pre-activated human mesenchymal stromal cells in combination with doxorubicin synergistically enhance tumor-suppressive activity in mice.

BACKGROUND AIMS: Previously, we showed that human mesenchymal stromal cells (hMSCs) were activated to express tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) upon TNF-α stimulation, induced cell death in triple-negative breast cancer (TNBC) MDA-MB-231 cells (MDA), and RNA released from apoptotic MDA further increased TRAIL expression in hMSCs. This feed-forward stimulation increased apoptosis in MDA cells. Here, we tested whether TRAIL-expressing hMSCs, in combination with a sub-toxic-dose of a chemotherapy drug doxorubicin, would overcome TRAIL resistance and create synergistic effects on targeting metastatic TNBC. METHODS: To optimize conditions for the combination treatment, we (i) selected an optimal condition to activate hMSCs for TRAIL expression, (ii) selected an optimal dose of doxorubicin treatment, (iii) examined underlying mechanisms in vitro and (iv) tested the efficacy of the optimized conditions in a xenograft mouse model of human breast cancer lung metastasis. RESULTS: The results showed that DNA fragments from apoptotic MDA triggered hMSCs to increase further TRAIL expression in an absent in melanoma 2 (AIM2)-dependent manner, and thus higher TRAIL-expressing hMSCs stimulated with synthetic DNA, poly(deoxyadenylic-deoxythymidylic) acid [poly(dA:dT)], more effectively suppressed tumor progression in vivo. Furthermore, activated hMSCs increased apoptosis in MDA cells when combined with a sub-toxic dose of doxorubicin, which was mediated by up-regulating TRAIL and Fas-related pathways. When we combined the optimized conditions, pre-activated hMSCs with poly (dA:dT) synergistically reduced tumor burden even with minimal doxorubicin treatment in a xenograft mouse model of human breast cancer lung metastasis. CONCLUSIONS: These results suggest that the treatment of hMSCs with a sub-toxic dose of doxorubicin can overcome TRAIL resistance and be a potential novel therapy for TNBC metastasis treatment.

MSCs derived from iPSCs with a modified protocol are tumor-tropic but have much less potential to promote tumors than bone marrow MSCs.

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFß, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for "off-the-shelf" therapies and bioengineering applications.

In Situ Recruitment of Human Bone Marrow-Derived Mesenchymal Stem Cells Using Chemokines for Articular Cartilage Regeneration.

Bone marrow-derived mesenchymal stem cells (BMSCs) are a good cell source for regeneration of cartilage as they can migrate directly to the site of cartilage injury and differentiate into articular chondrocytes. Articular cartilage defects do not heal completely due to the lack of chondrocytes or BMSCs at the site of injury. In this study, the chemotaxis of BMSCs toward chemokines, which may give rise to a complete regeneration of the articular cartilage, was investigated. CCR2, CCR4, CCR6, CXCR1, and CXCR2 were expressed in normal BMSCs and were increased significantly upon treatment with proinflammatory cytokines. BMSC migration was increased by MIP-3α and IL-8 more than by MCP-1 or SDF-1α. IL-8 and MIP-3α significantly enhanced the chemotaxis of BMSCs compared with MCP-1, SDF-1α, or PBS. Human BMSC recruitment to transplanted scaffolds containing either IL-8 or MIP-3α significantly increased in vivo compared to scaffolds containing PBS. Furthermore, IL-8- and MIP-3α-containing scaffolds enhanced tissue regeneration of an osteochondral defect site in beagle knee articular cartilage. Therefore, this study suggests that IL-8 and MIP-3α are the candidates that induce the regeneration of damaged articular cartilage.

Comparative analysis of gene expression in normal and degenerative human tendon cells: effects of cyclic strain.

BACKGROUND: Tendinopathy is a clinical problem for which treatment shows mixed results and treatment options are limited. Gene expression signatures early in the mechanotransduction pathway can accurately predict risk and correlate with different clinical outcomes. Studies aimed at elucidating the molecular mechanisms of tendinopathy have focused on small cohorts of genes that show an incomplete picture of the degeneration process. This study compared the effect of cyclic strain on gene expression in tendon cells from normal tendon and chronically painful areas of tendinopathy in 3 patients. METHODS: We measured a panel of mechanotransduction genes and cytoskeletal tensional balance with and without cyclic strain, which disrupts connective tissue synthetic-degradative equilibrium. Normal and degenerative tendons were obtained from patients undergoing surgery to treat chronic painful tendinopathy. A cyclic strain model was established to measure cytoskeletal tensional homeostasis. RESULTS: Prior to cyclic strain, the normal tendon cells exhibited varying patterns of elevated expression of 7 genes compared with degenerative tendon cells. In response to cyclic strain, gene expression of COL1A1, ITGA6, CTNNA1, and CLEC3B was up-regulated in normal tendon cells. Cyclic strain had no effect on degenerative tendon cells. Cyclic strain exacerbated the inhibition of protein synthesis in both cell types, especially in the degenerative tendon cells. CONCLUSION: Alterations in the pattern of gene expression are suggestive of a dynamic equilibrium between synthesis and degradation, whereby cell adhesion molecules are predominantly up-regulated to facilitate cellular reorientation in response to their altered functional environment. CLINICAL RELEVANCE: These data might have future applications, including the identification of markers for early diagnosis, targets for drug design, and indicators for treatment responsiveness and prognosis.

miR-449a regulates the chondrogenesis of human mesenchymal stem cells through direct targeting of lymphoid enhancer-binding factor-1.

microRNAs are small molecules, about 17-23 nucleotides in length, that act as translational regulators of their target gene. By binding to a target, microRNAs are known to either inhibit translation or induce degradation of the target. Despite the great interest in microRNAs, however, the exact targets of each individual microRNA in different processes remain largely unknown. In this study, we determined that the lymphoid enhancer-binding factor-1 (LEF-1) was expressed during the chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and sought to identify a novel microRNA targeting this gene. Through subsequent studies, we have identified, for the first time, one particular microRNA, miR-449a, that recognizes and regulates the expression of LEF-1 in a dose-dependent and sequence-specific manner. In addition, we observed that the inhibition of LEF-1 via miR-449a led to the subsequent repression of Sox 9, which is a well-established regulator of chondrogenesis. Collectively, this study demonstrated that miR-449a directly targets LEF-1, which in turn affects the expression of Sox 9, ultimately leading to the proper regulation of the differentiation and chondrogenesis of human MSCs (hBM-MSCs).

Identification and characterization of the promoter region of Kaposi's sarcoma-associated herpesvirus ORF11.

Open reading frame 11 (ORF11) of Kaposi's sarcoma-associated herpesvirus belongs to a herpesviral homologous protein family that is conserved among members of the gamma-herpesvirus subfamily. Little is known about the function of ORF11 and how this viral gene is regulated in KSHV life cycle. In this study, we have characterized the major transcript of the ORF11 gene, which is located adjacent and in the opposite orientation to a well-characterized viral IL-6 gene. Northern blot analysis revealed that the ORF11 gene is lytic viral gene with delayed-early expression kinetics. We have determined the 5' and 3' untranslated region of the unspliced ORF11 transcript and identified both the transcription start site and the transcription termination site. Core promoter region, representing ORF11 promoter activity, was mapped to a 160nt fragment 5' most proximal to the transcription start site. A functional TATA box was identified in the core promoter region. We also found that the characterized ORF11 gene promoter region is not responsive to Rta, the KSHV lytic switch protein. Our data help to elucidate transcription regulation of the KSHV ORF11 gene and to understand the biology of ORF11 in KSHV life cycle.

Histological and biomechanical properties of regenerated articular cartilage using chondrogenic bone marrow stromal cells with a PLGA scaffold in vivo.

The properties of regenerated cartilage using bone marrow-derived mesenchymal stem cells (MSCs) and poly lactic-co-glycolic acid (PLGA) scaffold composites pretreated with TGF-beta3 were investigated and compared to the non-TGF-beta3 treated MSCs/PLGA composites in a rabbit model. We prepared MSCs/PLGA scaffold composites and pretreated it with TGF-beta3 for 3 weeks prior to transplantation. Then, composites were transplanted to the osteochondral defect in the rabbit knee. After 12 weeks of transplantation, 10 of the 12 rabbits in which TGF-beta3 pretreated MSCs/PLGA scaffold composites were transplanted showed cartilaginous regeneration. In gross morphology, regenerated cartilage showed smooth, flush, and transparent features. In indentation test, this had about 80% of Young's modulus of normal articular cartilage. Histological examination demonstrated hyaline like cartilage structures with glycosaminoglycan and type II collagen expression. Histological scores were not statistically different to the normal articular cartilage. These results showed improvement of cartilage regeneration compared to the non-TGF-beta3 pretreated MSCs/PLGA scaffold composite transplanted group. Thus, we have successfully regenerated improved hyaline-like cartilage and determined the feasibility of treating damaged articular cartilage using MSCs/PLGA scaffold composite pretreated with TGF-beta3. Also, we suggest this treatment modality as another concept of cartilage tissue engineering.

ERK 1/2 activation in enhanced osteogenesis of human mesenchymal stem cells in poly(lactic-glycolic acid) by cyclic hydrostatic pressure.

The aim of this study was to identify the signal transduction pathways and mechano-transducers that play critical roles in the processes induced by changes in cyclic hydrostatic pressure and fluid shear in 3-dimensional (3D) culture systems. Mesenchymal stem cells were loaded into a polymeric scaffold and divided into three groups according to the stress treatment: static, fluid shear, and hydrostatic pressure with fluid shear. Cells were exposed daily to a hydrostatic pressure of 0.2 MPa for 1 min followed by 14 min rest with fluid flow at 30 rpm. Protein extracts were analyzed by Western blot for extracellular signal-regulated kinase 1/2 (ERK1/2). The complexes were cultured under the mechanical stimuli for 21 days with or without phospho-ERK1/2 inhibitor (U0126) and evaluated by RT-PCR, calcium contents, and immunohistochemistry. Under conditions of mechanical stimulation, the activation of ERK1/2 was sustained or increased with time. U0126 suppressed mechanical stimuli-induced expression of osteocalcin. In addition, calcium contents and the degrees of osteocalcin and osteopontin staining were decreased by this inhibitor. These results demonstrate that mechanical stimuli, particularly hydrostatic pressure with fluid shear, enhance osteogenesis in 3D culture systems via ERK1/2 activation.