Marrow-derived Precursor Cells Research Articles

40 Preclinical evaluation of a novel dendritic cell vaccine for kidney cancer

Abstract Background Dendritic cell (DC)-based vaccines have been previously shown to promote therapeutic T cell responses in both preclinical tumor models and in cancer patients, where extended patient overall survival has been noted in many cases. The goal of the present study is to develop a novel DC-based vaccine as an effective immuno-oncology (IO) agent for the prevention/treatment of kidney cancer. We previously demonstrated that actin-binding protein profilin1 (Pfn1) is overexpressed in tumor-associated vascular endothelial cells (ECs), where it may serve as a prognostic factor in human clear cell renal cancer (ccRCC). We further established a direct causal relationship between EC Pfn1 dysregulation, immune microenvironment alterations, and tumor progression in mouse models of RCC. The current work specifically explores whether Pfn1-targeted DC-based vaccines are effective in preventing orthotopic RCC in mice. Methods To generate Pfn1-targeted DC vaccines, we harvested and matured DCs from Balb/C mouse bone marrow-derived precursor cells in cultures containing rmIL4 and rmGM-CSF. DCs loaded with individual Pfn1 synthetic peptides (these sequences were identified using three MHC class I/II peptide-binding algorithms) were injected into syngeneic mice on days 0, 7 and 14. Spleens were harvested from immunized mice on day 21 to isolate CD4+ and CD8+ T cells, with T cells then stimulated with unmanipulated DCs or DCs pulsed with individual immunizing peptides to detect specific T cell activation, as measured by IFN-gamma release quantitated by ELISA. Tissue histology was performed to assess immune-related adverse events (iRAE) in vaccinated mice as a safety index. For cancer studies, Balb/c mice were immunized with DCs loaded with pooled Pfn1 peptides a few days prior to establishing either subcutaneous or orthotopic RCC tumors. Tumor-bearing mice were subjected to an identical booster vaccine prior one week later in advance of euthanasia and study end-point analyses. Results Our studies show that vaccination of Balb/c mice with DCs pulsed with Pfn1 peptides elicit specific CD4+ and/or CD8+ T cell responses without promoting irAEs. When applied in the prophylactic setting, DC-Pfn1 peptide vaccines substantially slow the growth of subcutaneous RCC tumors. In support of these findings, we have also developed preliminary data supporting the therapeutic effects of DC/Pfn1-peptide vaccines in a murine orthotopic model of RCC. Conclusions In summary, our studies provide first evidence for Pfn1-targeted IO agent in the cancer setting. Given that the survival benefit for dual immune-check point inhibitors (ICI)/anti-angiogenic therapy remains modest and is limited to a small minority of ccRCC patients, Pfn1-targeted vaccines could represent a novel therapeutic agent for improved patient outcomes when applied alone or in combination with in-clinic ICI agents. DOD CDMRP Funding: yes

Cortactin controls bone homeostasis through regulating the differentiation of osteoblasts and osteoclasts.

Cortactin (CTTN), a cytoskeletal protein and substrate of Src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that CTTN was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that CTTN promoted the in vitro differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, CTTN was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that CTTN could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by CTTN siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by CTTN overexpression. Notably, transplantation of CTTN-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, CTTN-silenced BMSCs expressed higher levels of receptor activator of nuclear factor κB ligand (RANKL) than control BMSCs did and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that CTTN favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of CTTN expression may be advantageous for maintaining bone homeostasis.

In situ co-deposition synthesis for collagen-Astragalus polysaccharide composite with intrafibrillar mineralization as potential biomimetic-bone repair materials.

A hybrid material possessing both componential and structural imitation of bone tissue is the preferable composites for bone defect repair. Inspired by the microarchitecture of native bone, this work synthesized in vitro a functional mineralized collagen fibril (MCF) material by utilizing the method of in situ co-precipitation, which was designed to proceed in the presence of Astragalus polysaccharide (APS), thus achieving APS load within the biomineralized collagen-Astragalus polysaccharide (MCAPS) fibrils. Transmission electron microscope (TEM), selected area electron diffraction (SAED) and scanning electronic microscopy (SEM) identified the details of the intrafibrillar mineralization of the MCAPS fibrils, almost mimicking the secondary level of bone tissue microstructure. A relatively uniform and continuous mineral layer formed on and within all collagen fibrils and the mineral phase was identified as typical weak-crystalline hydroxyapatite (HA) with a Ca/P ratio of about 1.53. The proliferation of bone marrow-derived mesenchymal stem cells (BMSC) and mouse embryo osteoblast precursor cells (MC3T3-E1) obtained a significant promotion by MCAPS. As for the osteogenic properties of MCAPS, a distinct increase in the alkaline phosphatase (ALP) activity and the number of calcium nodules (CN) in BMSC and MC3T3-E1 was detected. The up-regulation of three osteogenic-related genes of RUNX-2, BMP-2 and OCN were confirmed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to further verify the osteogenic performance promotion of MCAPS. A period of 14 days of culture demonstrated that MCAPS-L exhibited a preferable efficacy in enhancing ALP activity and CN quantity, as well as in promoting the expression of osteogenic-related genes over MCAPS-M and MCAPS-H, indicating that a lower dose of APS within the material of MCAPS is more appropriate for its osteogenesis promotion properties.

Incomplete response to Anti-VEGF therapy in neovascular AMD: Exploring disease mechanisms and therapeutic opportunities.

Intravitreal anti-vascular endothelial growth factor (VEGF) drugs have revolutionized the treatment of neovascular age-related macular degeneration (NVAMD). However, many patients suffer from incomplete response to anti-VEGF therapy (IRT), which is defined as (1) persistent (plasma) fluid exudation; (2) unresolved or new hemorrhage; (3) progressive lesion fibrosis; and/or (4) suboptimal vision recovery. The first three of these collectively comprise the problem of persistent disease activity (PDA) in spite of anti-VEGF therapy. Meanwhile, the problem of suboptimal vision recovery (SVR) is defined as a failure to achieve excellent functional visual acuity of 20/40 or better in spite of sufficient anti-VEGF treatment. Thus, incomplete response to anti-VEGF therapy, and specifically PDA and SVR, represent significant clinical unmet needs. In this review, we will explore PDA and SVR in NVAMD, characterizing the clinical manifestations and exploring the pathobiology of each. We will demonstrate that PDA occurs most frequently in NVAMD patients who develop high-flow CNV lesions with arteriolarization, in contrast to patients with capillary CNV who are highly responsive to anti-VEGF therapy. We will review investigations of experimental CNV and demonstrate that both types of CNV can be modeled in mice. We will present and consider a provocative hypothesis: formation of arteriolar CNV occurs via a distinct pathobiology, termed neovascular remodeling (NVR), wherein blood-derived macrophages infiltrate the incipient CNV lesion, recruit bone marrow-derived mesenchymal precursor cells (MPCs) from the circulation, and activate MPCs to become vascular smooth muscle cells (VSMCs) and myofibroblasts, driving the development of high-flow CNV with arteriolarization and perivascular fibrosis. In considering SVR, we will discuss the concept that limited or poor vision in spite of anti-VEGF may not be caused simply by photoreceptor degeneration but instead may be associated with photoreceptor synaptic dysfunction in the neurosensory retina overlying CNV, triggered by infiltrating blood-derived macrophages and mediated by Müller cell activation Finally, for each of PDA and SVR, we will discuss current approaches to disease management and treatment and consider novel avenues for potential future therapies.

Bovine Milk Extracellular Vesicles Are Osteoprotective by Increasing Osteocyte Numbers and Targeting RANKL/OPG System in Experimental Models of Bone Loss

Studying effects of milk components on bone may have a clinical impact as milk is highly associated with bone maintenance, and clinical studies provided controversial associations with dairy consumption. We aimed to evaluate the impact of milk extracellular vesicles (mEVs) on the dynamics of bone loss in mice. MEVs are nanoparticles containing proteins, mRNA and microRNA, and were supplemented into the drinking water of mice, either receiving diet-induced obesity or ovariectomy (OVX). Mice receiving mEVs were protected from the bone loss caused by diet-induced obesity. In a more severe model of bone loss, OVX, higher osteoclast numbers in the femur were found, which were lowered by mEV treatment. Additionally, the osteoclastogenic potential of bone marrow-derived precursor cells was lowered in mEV-treated mice. The reduced stiffness in the femur of OVX mice was consequently reversed by mEV treatment, accompanied by improvement in the bone microarchitecture. In general, the RANKL/OPG ratio increased systemically and locally in both models and was rescued by mEV treatment. The number of osteocytes, as primary regulators of the RANKL/OPG system, raised in the femur of the OVX mEVs-treated group compared to OVX non-treated mice. Also, the osteocyte cell line treated with mEVs demonstrated a lowered RANKL/OPG ratio. Thus, mEVs showed systemic and local osteoprotective properties in two mouse models of bone loss reflected in reduced osteoclast presence. Data reveal mEV potential in bone modulation, acting via osteocyte enhancement and RANKL/OPG regulation. We suggest that mEVs could be a therapeutic candidate for the treatment of bone loss.

The glycosyltransferase EXTL2 promotes proteoglycan deposition and injurious neuroinflammation following demyelination

BackgroundChondroitin sulfate proteoglycans (CSPGs) are potent inhibitors of axonal regrowth and remyelination. More recently, they have also been highlighted as a modulator of macrophage infiltration into the central nervous system in experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis.MethodsWe interrogated results from single nucleotide polymorphisms (SNPs) lying in or close to genes regulating CSPG metabolism in the summary results from two publicly available systematic studies of multiple sclerosis (MS) genetics. A demyelinating injury model in the spinal cord of exostosin-like 2 deficient (EXTL2-/-) mice was used to investigate the effects of dysregulation of EXTL2 on remyelination. Cell cultures of bone marrow-derived macrophages and primary oligodendrocyte precursor cells and neurons were supplemented with purified CSPGs or conditioned media to assess potential mechanisms of action.ResultsThe strongest evidence for genetic association was seen for SNPs mapping to the region containing the glycosyltransferase exostosin-like 2 (EXTL2), an enzyme that normally suppresses CSPG biosynthesis. Six of these SNPs showed genome-wide significant evidence for association in one of the studies with concordant and nominally significant effects in the second study. We then went on to show that a demyelinating injury to the spinal cord of EXTL2−/− mice resulted in excessive deposition of CSPGs in the lesion area. EXTL2−/− mice had exacerbated axonal damage and myelin disruption relative to wild-type mice, and increased representation of microglia/macrophages within lesions. In tissue culture, activated bone marrow-derived macrophages from EXTL2−/− mice overproduce tumor necrosis factor α (TNFα) and matrix metalloproteinases (MMPs).ConclusionsThese results emphasize CSPGs as a prominent modulator of neuroinflammation and they highlight CSPGs accumulating in lesions in promoting axonal injury.

Inhibitory effect of Chaenomelis Fructus ethanol extract on receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenesis

The fruit of Chaenomeles sinensis (Thouin) Koehne (Chaenomelis Fructus) known as “Mo-Gua” in Korea has been commonly used in traditional medicine to treat inflammatory diseases, such as sore throat. However, its effect on bone metabolism has not been elucidated yet. Here, we examined the effect of Chaenomelis Fructus ethanol extract (CFE) on receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation and formation. CF-E considerably inhibited osteoclast differentiation and tartrate-resistant acid phosphatase-positive multinuclear cell formation from bone marrow-derived macrophages and osteoclast precursor cells in a dose-dependent manner. In addition, the formation of actin rings and resorption pits were significantly suppressed in CF-E-treated osteoclasts as compared with the findings in non-treated control cells. Consistent with these phenotypic inhibitory results, the expressions of osteoclast differentiation marker genes (Acp5, Atp6v0d2 , Oscar, CtsK, and Tm7sf4) and Nfatc1 , a pivotal transcription factor for osteoclastogenesis, were markedly decreased by CF-E treatment. The inhibitory effect of CF-E on RANKL-induced osteoclastogenesis was associated with the suppression of NFATc1 expression, not by regulation of mitogen-activated protein kinases and NF-κB activation but by the inactivation of phospholipase C gamma 1 and 2. These results indicate that CF-E has an inhibitory effect on osteoclast differentiation and formation, and they suggest the possibility of CF-E as a traditional therapeutic agent against bone-resorptive diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis.

Blockade of myeloid-derived suppressor cell function by valproic acid enhanced anti-PD-L1 tumor immunotherapy

Regardless of the remarkable clinical success of immune checkpoint blockade (ICB) against PD-1/PD-L1 pathway, this approach has encountered drawbacks in most patients due to the activation of tumor immunosuppressive factors such as myeloid-derived suppressor cells (MDSCs). Histone deacetylase (HDAC) inhibitors combat ICB resistance by attenuating the immunosuppressive function of MDSCs and increasing PD-L1 expression on tumor cells. However, whether an HDAC inhibitor - valproic acid (VPA) suppression of MDSCs function could enhance PD-L1 blockade-mediated tumor immunotherapy remains unknown. Here we report that VPA and anti-PD-L1 antibody combined treatment promoted the polarization of bone marrow-derived precursor cells into M-MDSCs. Interestingly, the combination treatment of VPA and anti-PD-L1 antibody activated IRF1/IRF8 transcriptional axis in MDSCs leading to blockade of their immunosuppressive function by downregulating the expression of IL-10, IL-6, and ARG1 while re-activating CD8+ T-cells for the production of TNFα to further enhance anti-tumor immunity. These observations provide further rationale for the combination therapy of VPA with anti-PD-L1 antibody in preclinical settings.

On the road to new treatments for multiple sclerosis: targeting dendritic cell migration into the central nervous system.

On the road to new treatments for multiple sclerosis: targeting dendritic cell migration into the central nervous system.

HMGB1 Causes Anemia of Inflammation By Modulating Erythropoietin Signal Transduction

The mechanisms underlying the development of erythropoietin (EPO)-refractory anemia in the setting of chronic inflammatory states are largely unknown. Elevated levels of the classical inflammatory mediators decrease red cell output. However, pathologic concentrations of many of these molecules do not persist beyond the acute phase, indicating that specific mediators are likely to play a role in the anemia associated with chronic inflammation. High mobility group box protein 1 (HMGB1) is a potent alarmin able to induce tissue injury during the acute and chronic phases of inflammation, and recently, shown to contribute to anemia in a murine model of sepsis. Here, we show that HMGB1 directly inhibits erythropoiesis by modulating EPO signal transduction in human erythroid cells through a newly identified HMGB1 receptor, which is surprisingly the erythropoietin receptor (EPOR). Surface plasmon resonance (SPR) reveals that HMGB1 binds the extracellular domain of EPOR (Kd = 130nM) with an affinity comparable to that of EPO. Cysteine residues contained within the A- and B-box domains of HMGB1 that have previously been shown to mediate HMGB1-receptor interactions are also responsible for the EPOR-HMGB1 interaction since a mutant form of HMGB1 lacking these cysteine residues (i.e. 3S HMGB1) fails to bind the EPOR. Cell-based assays suggest that the direct binding of HMGB1 to the EPOR and the subsequent degradation of EPOR accounts for altered EPO signaling by HMGB1. Biologically, HMGB1 reduces the phosphorylation of intracellular EPO effectors including JAK2 (2-fold reduction), STAT5 (4-fold), and ERK1/2 (4-fold). Decreased effector phosphorylation is not due to the increased activity of SHP1/2 phosphatases further implicating inhibition at the receptor level. Loss of EPO signaling due to HMGB1 binding results in decreased erythroid proliferation of differentiated CD34+ cells at the EPO-dependent stages of erythropoiesis: Day 14: 1.03x108 ± 4.67x107 cells/mL vs 1.87x106 ± 9.70x105 cells/mL, vehicle vs HMGB1, respectively. In addition, HMGB1 decreases the numbers of colony forming unit-erythroid (CFU-E) progenitors by 60%, and these progenitors fail to undergo terminal erythroid differentiation with a block at the basophilic erythroblast stage and apoptosis of late-stage erythroblasts as determined by flow cytometric analysis of annexin V staining. To understand the consequences of HMGB1-EPOR interactions on the EPO-induced transcriptome, RNA-sequencing was performed on purified human CFU-E dosed with HMGB1 and EPO. HMGB1 reduces the expression of known EPO target genes (ERFE, CISH, EGR1), and concomitantly, upregulates a number of unique transcripts (ETS2, VMP1, NFKBIZ) suggesting that HMGB1-EPOR interactions may alter receptor conformation in manner that differentially activates the EPOR and consequently, gene expression. Finally, in a mouse model of sepsis survival, bone marrow-derived erythroid precursor cells contain diminished phosphorylated STAT5 levels at a time when elevated HMGB1 plasma concentrations are observed, thereby demonstrating that the loss of EPO signal transduction also occurs in vivo. Taken together, our work identifies HMGB1 as a novel inhibitor of EPO signaling through its interaction with the EPOR, and strongly implicates HMGB1 as a previously undiscovered effector of EPO-refractory anemia associated with chronic inflammation. DisclosuresNo relevant conflicts of interest to declare.

Aryl hydrocarbon receptor activation by benzo(a)pyrene inhibits proliferation of myeloid precursor cells and alters the differentiation state as well as the functional phenotype of murine bone marrow-derived macrophages

Intensive research during the past decade has highlighted the impact of the regulatory function of the aryl hydrocarbon receptor (AhR) in immunity. In this study, we focused on the influence of AhR activation on the differentiation of murine bone marrow-derived myeloid precursor cells into mature macrophages. Our results show that the activation of AhR by subtoxic doses of the AhR ligand benzo(a)pyrene (BaP) impaired the proliferation of bone marrow cells (BMCs) whereas the proportion of resulting adherent cells was not affected. Flow cytometric analysis revealed that the number of mature bone marrow-derived macrophages (BMMs) was significantly decreased by AhR activation. However, expression of the murine macrophage marker F4/80, the major histocompatibility complex class II (MHC-II) and the Fcγ receptor I (FcγRI/CD64) were upregulated on BaP-exposed BMMs in an AhR-dependent manner. Analysis of cytokine secretion after BMM activation with heat-killed (hk) salmonellae showed that BaP exposure resulted in suppressed secretion of interleukin (IL)-1β, IL-6 and the chemokine CXC motif ligand 1 (CXCL1). In contrast, the release of tumor necrosis factor (TNF)-α and IL-10 was increased following BaP exposure. In addition, the production of antimicrobial nitric oxide (NO) was increased AhR-dependently. Bacterial stimulation of BaP exposed BMMs also induced the expression of MHC-II and CD64, while the expression of F4/80 was dramatically decreased. In summary, this study demonstrates for the first time that sustained exposure over 6 days of bone marrow-derived myeloid precursors to subtoxic doses of BaP critically interferes with differentiation and activation of BMMs. We could convincingly show that AhR-induced gene regulation is crucial for homeostasis of pro- and anti-inflammatory cytokines during macrophage activation.

The tumour suppressor CDKN2A/p16INK4a regulates adipogenesis and bone marrow-dependent development of perivascular adipose tissue.

The genomic CDKN2A/B locus, encoding p16INK4a among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16INK4a is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16INK4a knock-down in 3T3/L1 preadipocytes or p16INK4a deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16INK4a in adipose tissue formation. p16INK4a-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16INK4a-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16INK4a-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16INK4a gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16INK4a modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

Milk-Derived Nanoparticle Fraction Promotes the Formation of Small Osteoclasts But Reduces Bone Resorption.

The general consensus is that milk promotes bone growth and density because is a source of calcium and contains components that enhance intestinal calcium uptake or directly affect bone metabolism. In this study, we investigated the effect of bovine-derived milk 100,000 g pellet (P100), which contains nanoparticles (<220 nm) including extracellular vesicles, on osteoclast differentiation and bone resorption. Bone marrow-derived osteoclast precursor cells were differentiated into osteoclasts by M-CSF and RANKL (control) and in the presence of milk P100. Milk P100 treatment until day 4 increased the number of TRAP-positive mononuclear cells and small (≤5 nuclei) osteoclasts. The number of large (≥6 nuclei) osteoclasts remained the same. These alterations were associated with increased expression of TRAP, NFATc1, and c-Fos. Cells seeded in a calcium-phosphate coated plate or bone slices showed reduced resorption area when exposed to milk P100 during the differentiation phase and even after osteoclast formation. Interestingly, milk P100 treatment enhanced Cathepsin K expression but reduced Carbonic Anhydrase 2 gene expression. Moreover, intracellular acid production was also decreased by milk P100 treatment. Oral delivery of milk P100 to female DBA1/J mice for 7 weeks did not alter bone area; however, increased osteoclast number and area in tibia without changes in serum RANKL and CTX-I levels. We showed for the first time the effect of milk P100 on osteoclast differentiation both in vitro and in vivo and found that milk P100 increased the formation of small osteoclasts but this does not lead to more bone resorption probably due to reduced acid secretion. J. Cell. Physiol. 232: 225-233, 2017. © 2016 Wiley Periodicals, Inc.

CCR5 facilitates endothelial progenitor cell recruitment and promotes the stabilization of atherosclerotic plaques in ApoE-/- mice.

IntroductionUnstable atherosclerotic plaques are prone to rupture, which leads to atherothrombosis. Endothelial progenitor cells (EPCs) are bone marrow-derived precursor cells that may repair vascular injury in atherosclerosis. Chemokine (C-C motif) receptor 5 (CCR5) promotes mobilization of EPCs. In this study, we investigated the therapeutic potential of CCR5-overexpressing EPCs on plaque stabilization in an apolipoprotein E (ApoE)−/− mouse model.MethodsThe expression of CCR5 and its cognate ligand chemokine (C-C motif) ligand 5 (CCL5) was examined in atherosclerotic aortas of humans and mice by immunohistochemistry. Splenectomized ApoE−/− C57BL/6 J mice fed a high-fat diet for 24 weeks were intravenously injected with EPCs transfected with CCR5 overexpression lentivirus. The recruitment of EPCs over the atherosclerotic plaques was evaluated by immunofluorescence. The content of lipid, smooth muscle cells, monocytes/macrophages, and endothelial cells in atherosclerotic plaques was assayed by specific immunostaining. The serum levels of atherosclerosis-related inflammatory factors in ApoE−/− mice were measured by mouse atherosclerosis antibody array I.ResultsCCR5 and CCL5 are highly expressed in atherosclerotic plaques in both humans and mice. The ApoE−/− mice with CCR5-overexpressing EPC treatment demonstrated a more stable plaque formation with enhanced recruitment of EPC, reduced lipid, and macrophage content in the atherosclerotic plaques. CCR5-overexpressing EPC treatment also increased the content of endothelial cells and nitric oxide production in the plaques. In addition, the serum levels of interleukin-3 (IL-3), IL-5, IL-6, IL-13, CD40, and tumor necrosis factor-alpha and the plaque contents of IL-6 and matrix metalloproteinase-9 were reduced in mice with CCR5-overexpressing EPC treatment.ConclusionsThese findings suggest that CCR5 is a novel therapeutic target in EPC treatment for stabilization of atherosclerotic plaques.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0026-0) contains supplementary material, which is available to authorized users.

TGFβ and PDGF-B signaling blockade inhibits myofibroblast development from both bone marrow-derived and keratocyte-derived precursor cells in vivo

Myofibroblasts, the primary cells associated with corneal stromal haze (opacity), can be derived from both cornea-derived and bone marrow-derived precursor cells. In the present study, the role of TGFβ or PDGF blockage on bone marrow-derived myofibroblast development was investigated using a green fluorescent protein (GFP) chimeric bone marrow mouse model and plasmid vectors that blocked TGFβ or PDGF signaling. At the peak of corneal haze one month after irregular phototherapeutic keratectomy the central stroma had significantly less alpha-smooth muscle actin (α-SMA)-positive cells derived from GFP+ bone marrow-derived cells or GFP− keratocyte/corneal fibroblast-derived cells when corneas were treated with the TGFβ blocking vector pGFPC1.TGFRBKDEL or the PDGF blocking vector pCMV.PDGFRB.23KDEL compared with the corresponding empty vector treated or untreated control groups. In individual animals, 30–60% of myofibroblasts were derived from bone marrow-derived precursor cells and 40–70% of myofibroblasts were derived from keratocyte-derived precursor cells. TGFβ and PDGF regulate corneal myofibroblast development from bone marrow-derived precursor cells and keratocyte/corneal fibroblast-derived precursor cells.

Platelet-derived growth factor D: A new player in the complex cross-talk between cholangiocarcinoma cells and cancer-associated fibroblasts

Platelet-derived growth factor D: A new player in the complex cross-talk between cholangiocarcinoma cells and cancer-associated fibroblasts

The Role of CSF1 in Hepatocellular Carcinoma‐Recruited Macrophages

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and it is clear that local tissue microenvironment plays a key role in its progression. Macrophages are suspected to effect tumor expansion and malignancy by promoting inflammatory responses and tissue remodeling. Colony stimulating factor (CSF1) has been shown to promote macrophage differentiation, survival and proliferation. Although CSF1 is highly expressed in liver, little is known about how CSF signaling impacts macrophages in HCC. We found that CSFR positive macrophages are located in hyperplastic non‐tumor and peri‐tumoral regions of a chemically induced murine HCC model. This expression mimics that seen in HCC specimens from human patients with a poor prognosis. We hypothesized that CSF1 derived from HCC cells promotes macrophage differentiation and viability. Murine bone marrow derived precursor cells were differentiated into macrophages using HCC conditioned culture media. Macrophages treated with a colony stimulating receptor small molecule inhibitor (CSFRi) exhibited altered morphology and decreased proliferation in vitro. Small molecule mediated inhibition of CSFR promoted a shift in macrophage polarization from M2 (pro‐tumor) to M1 (anti‐tumor). These findings suggest a critical role for HCC‐cell mediated CSF signaling in macrophage proliferation, viability, and polarization.

An Adipoinductive Role of Inflammation in Adipose Tissue Engineering: Key Factors in the Early Development of Engineered Soft Tissues

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1β are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.

Endothelial Progenitor Cells: A Novel Cell-based Therapy in Orthopaedic Surgery

Endothelial progenitor cells (EPCs) can be defined as bone marrow-derived precursor cells with the ability to differentiate into endothelial cells and to participate in the formation of new blood vessels. Bone marrow is the primary source of endothelial progenitors, which can be mobilized to the peripheral circulation and may seed in remote organs and tissues such as liver, spleen, heart, muscle, and adipose tissue. EPCs have been shown to express various endothelial surface markers, such as CD34, VEGFR2, and CD133, and to home to sites of ischemia1 (Figure 1).Figure 1: Phenotype of cultured endothelial progenitor cells (EPCs) can be characterized according to their ability to uptake Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) to confirm their endothelial lineage. Image demonstrates Dil-Ac-LDL-stained EPCs under fluorescent microscope (×40).Vascular ingrowth at the fracture site has a cardinal role in the healing process and regeneration of the bone following fracture. However, segmental bone defects after severe trauma, infection, and surgical removal of tumors remain a major clinical problem. These facts, together with reports documenting the remarkable therapeutic potential of EPCs to improve neovascularization and tissue perfusion in other disciplines, have been the major drivers recently to investigate the effects of EPC therapy on fracture healing. Matsumoto et al2 investigated the therapeutic potential of systemically administered CD34+ cells on fracture healing in a rodent model. The authors transplanted human peripheral blood CD34+ cells, mononuclear cells, or saline into immunodeficient rats with a non-healing femoral fracture. Fracture healing was significantly enhanced in the CD34+ group compared with the mononuclear cell and saline groups. Laser Doppler imaging demonstrated that fracture-induced ischemia was significantly reduced in the CD34+ cell-transplanted group compared with the other groups. The authors also noted that approximately 20% of human peripheral blood CD34+ cells expressed mRNA for osteocalcin after transplantation to a fracture site; this outcome may indicate the potential of CD34+ cells for osteogenic and endothelial differentiation. Atesok et al3 evaluated the effects of the local use of ex vivo-expanded EPCs on the stimulation of angiogenesis and the promotion of bone healing at a fracture site in a rat femur osteotomy model. Based on the results of radiographic, histologic, and micro-CT comparisons of the EPC-treated group with a control group, the authors stated that “local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.” In a similar study, the same research group reported that local EPC therapy favorably affects biomechanical stability.4 Recent reports also suggest that EPCs derived from peripheral blood contribute to osteogenic differentiation by mesenchymal stem cells (MSCs) in vitro and that MSCs support the proliferation of EPCs.5 Aguirre et al6 investigated the interactions between bone marrow EPCs (BM-EPCs) and MSCs in an in vitro co-culture system. Their data suggested that cross-talk occurs between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms, leading to modulation of the angiogenic response. In a rat model study, Seebach et al7 observed a synergistic effect between EPCs and MSCs and suggested that the initial stage of neovascularization by EPCs be considered crucial for complete bone regeneration. Positive effects of EPC therapy have been demonstrated in ligament tissue regeneration, as well. Matsumoto et al8 recently demonstrated that CD34- and CD146-expressing vascular cells exist in human anterior cruciate ligament tissues, have a potential for multilineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured anterior cruciate ligament. In a rodent model, Tei et al9 studied the effects of locally transplanted human peripheral blood CD34+ cells on the healing of medial collateral ligament injury. Macroscopic, histologic, and biomechanical assessments showed significantly enhanced ligament healing in a CD34+ cell transplantation group compared with a control group. The authors suggested that “local transplantation of circulating human CD34+ cells may augment the ligament healing process by promoting a favorable environment through neovascularization.” Based on the promising results from ex vivo and animal model EPC studies, clinical trials have been started. As a pilot case from a clinical trial, Kuroda et al10 reported the results of transplantation of autologous peripheral blood CD34+ cells—the hematopoietic/EPC-enriched population—into a patient with nonunion of a tibia fracture. Clinical and radiologic healing of the fracture was achieved at 12 weeks after the cell therapy with bone grafting, and no serious short-term complications were encountered. EPCs, with their unique features, such as the ability to differentiate into endothelial cells and to participate in the establishment of neovasculature, in addition to their high plasticity, may offer therapeutic alternatives for both bone and ligament tissue engineering. It is likely that more investigators will be attracted in the near future to exploring the potential of these cells in orthopaedic surgery.11

Endothelial Progenitor Cells: A Novel Cell-based Therapy in Orthopaedic Surgery

Endothelial progenitor cells (EPCs) can be defined as bone marrow-derived precursor cells with the ability to differentiate into endothelial cells and to participate in the formation of new blood vessels. Bone marrow is the primary source of endothelial progenitors, which can be mobilized to the peripheral circulation and may seed in remote organs and tissues such as liver, spleen, heart, muscle, and adipose tissue. EPCs have been shown to express various endothelial surface markers, such as CD34, VEGFR2, and CD133, and to home to sites of ischemia1 (Figure 1).Figure 1: Phenotype of cultured endothelial progenitor cells (EPCs) can be characterized according to their ability to uptake Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) to confirm their endothelial lineage. Image demonstrates Dil-Ac-LDL-stained EPCs under fluorescent microscope (×40).Vascular ingrowth at the fracture site has a cardinal role in the healing process and regeneration of the bone following fracture. However, segmental bone defects after severe trauma, infection, and surgical removal of tumors remain a major clinical problem. These facts, together with reports documenting the remarkable therapeutic potential of EPCs to improve neovascularization and tissue perfusion in other disciplines, have been the major drivers recently to investigate the effects of EPC therapy on fracture healing. Matsumoto et al2 investigated the therapeutic potential of systemically administered CD34+ cells on fracture healing in a rodent model. The authors transplanted human peripheral blood CD34+ cells, mononuclear cells, or saline into immunodeficient rats with a non-healing femoral fracture. Fracture healing was significantly enhanced in the CD34+ group compared with the mononuclear cell and saline groups. Laser Doppler imaging demonstrated that fracture-induced ischemia was significantly reduced in the CD34+ cell-transplanted group compared with the other groups. The authors also noted that approximately 20% of human peripheral blood CD34+ cells expressed mRNA for osteocalcin after transplantation to a fracture site; this outcome may indicate the potential of CD34+ cells for osteogenic and endothelial differentiation. Atesok et al3 evaluated the effects of the local use of ex vivo-expanded EPCs on the stimulation of angiogenesis and the promotion of bone healing at a fracture site in a rat femur osteotomy model. Based on the results of radiographic, histologic, and micro-CT comparisons of the EPC-treated group with a control group, the authors stated that “local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.” In a similar study, the same research group reported that local EPC therapy favorably affects biomechanical stability.4 Recent reports also suggest that EPCs derived from peripheral blood contribute to osteogenic differentiation by mesenchymal stem cells (MSCs) in vitro and that MSCs support the proliferation of EPCs.5 Aguirre et al6 investigated the interactions between bone marrow EPCs (BM-EPCs) and MSCs in an in vitro co-culture system. Their data suggested that cross-talk occurs between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms, leading to modulation of the angiogenic response. In a rat model study, Seebach et al7 observed a synergistic effect between EPCs and MSCs and suggested that the initial stage of neovascularization by EPCs be considered crucial for complete bone regeneration. Positive effects of EPC therapy have been demonstrated in ligament tissue regeneration, as well. Matsumoto et al8 recently demonstrated that CD34- and CD146-expressing vascular cells exist in human anterior cruciate ligament tissues, have a potential for multilineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured anterior cruciate ligament. In a rodent model, Tei et al9 studied the effects of locally transplanted human peripheral blood CD34+ cells on the healing of medial collateral ligament injury. Macroscopic, histologic, and biomechanical assessments showed significantly enhanced ligament healing in a CD34+ cell transplantation group compared with a control group. The authors suggested that “local transplantation of circulating human CD34+ cells may augment the ligament healing process by promoting a favorable environment through neovascularization.” Based on the promising results from ex vivo and animal model EPC studies, clinical trials have been started. As a pilot case from a clinical trial, Kuroda et al10 reported the results of transplantation of autologous peripheral blood CD34+ cells—the hematopoietic/EPC-enriched population—into a patient with nonunion of a tibia fracture. Clinical and radiologic healing of the fracture was achieved at 12 weeks after the cell therapy with bone grafting, and no serious short-term complications were encountered. EPCs, with their unique features, such as the ability to differentiate into endothelial cells and to participate in the establishment of neovasculature, in addition to their high plasticity, may offer therapeutic alternatives for both bone and ligament tissue engineering. It is likely that more investigators will be attracted in the near future to exploring the potential of these cells in orthopaedic surgery.11