Rama Garimella

Role of extracellular membrane vesicles in the pathogenesis of various diseases, including cancer, renal diseases, atherosclerosis, and arthritis.

Extracellular membrane vesicles (MVs) 30–1000 nm in diameter and of varying cellular origins are increasingly recognized for their participation in a range of processes, including the pathogenesis of various diseases, such as: (1) atherosclerosis, (2) thromboembolism, (3) osteoarthritis (OA), (4) chronic renal disease and pulmonary hypertension, (5) tissue invasion and metastasis by cancer cells, (6) gastric ulcers and bacterial infections, and (7) periodontitis. MVs are derived from many different cell types and intracellular mechanisms, and perform different metabolic functions or roles, depending on the cell of origin.The presence of a metabolically active, outer membrane is a distinguishing feature of all MVs, regardless of their cell type of origin and irrespective of terminologies applied to them such as exosomes, microparticles, or matrix vesicles. The MV membrane provides one of the few protected and controlled internal microenvironments outside cells in which specific metabolic objectives of the host cell may be pursued vigorously at a distance from the host cell. MVs are also involved in various forms of normal and abnormal intercellular communication. Evidence is emerging that circulating MVs are good predictors of the severity of several diseases. In addition, recently, the role of MVs in inducing immunity against cancer cells and bacterial infections has become a topic of interest to researchers in the area of therapeutics. The main objective of this review is to list and briefly describe the increasingly well-defined roles of MVs in selected diseases in which they seem to have a significant role in pathogenesis.

Sustained Osteomalacia of Long Bones Despite Major Improvement in Other Hypophosphatasia- Related Mineral Deficits in Tissue Nonspecific Alkaline Phosphatase/Nucleotide Pyrophosphatase Phosphodiesterase 1 Double-Deficient Mice

We have shown previously that the hypomineralization defects of the calvarium and vertebrae of tissue nonspecific alkaline phosphatase (TNAP)-deficient (Akp2-/-) hypophosphatasia mice are rescued by simultaneous deletion of the Enpp1 gene, which encodes nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Conversely, the hyperossification in the vertebral apophyses typical of Enpp1-/- mice is corrected in [Akp2-/-; Enpp1-/-] double-knockout mice. Here we have examined the appendicular skeletons of Akp2-/-, Enpp1-/-, and [Akp2-/-; Enpp1-/-] mice to ascertain the degree of rescue afforded at these skeletal sites. Alizarin red and Alcian blue whole mount analysis of the skeletons from wild-type, Akp2-/-, and [Akp2-/-; Enpp1-/-] mice revealed that although calvarium and vertebrae of double-knockout mice were normalized with respect to mineral deposition, the femur and tibia were not. Using several different methodologies, we found reduced mineralization not only in Akp2-/- but also in Enpp1-/- and [Akp2-/-; Enpp1-/-] femurs and tibias. Analysis of calvarial- and bone marrow-derived osteoblasts for mineralized nodule formation in vitro showed increased mineral deposition by Enpp1-/- calvarial osteoblasts but decreased mineral deposition by Enpp1-/- long bone marrow-derived osteoblasts in comparison to wild-type cells. Thus, the osteomalacia of Akp2-/- mice and the hypomineralized phenotype of the long bones of Enpp1-/- mice are not rescued by simultaneous deletion of TNAP and NPP1 functions.

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

Skeletal remodeling is a finely orchestrated process coupling bone formation to bone resorption. The dynamics of coupling is regulated by the microenvironment at the bone remodeling site, which in turn is influenced by the intercellular communication between cells like osteoclasts and osteoblasts. Understanding the dynamics of coupling is important in devising new therapeutic approaches to the treatment of skeletal diseases characterized by disturbances in the bone remodeling process. In this study, we report the localization of bone morphogenetic proteins (BMPs) in osteoclasts generated from primary cocultures of bone marrow cells from mouse femur and tibia with mouse calvarial osteoblasts, using immunocytochemistry and in situ hybridization. Positive staining was seen in osteoclasts for BMP-2, -4, -6, and -7. Real-time PCR was used to quantitatively confirm the expression of transcripts for BMP-2, BMP-4, and BMP-6 mRNA in murine osteoclasts. Finally, the presence of BMP-2, -4, -6, and-7 proteins was confirmed in osteoclast lysates by Western blotting. Overall, our data suggest a possible direct role for osteoclasts in promoting bone formation via expression and synthesis of BMPs, which then would play an important role in promoting the recruitment, proliferation, and differentiation of osteoblasts at bone resorption sites.

Matrix vesicles are carriers of bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), and noncollagenous matrix proteins

Matrix vesicles (MVs) are well positioned in the growth plate to serve as a carrier of morphogenetic information to nearby chondrocytes and osteoblasts. Bone morphogenetic proteins (BMPs) carried in MVs could promote differentiation of these skeletal cells. Vascular endothelial growth factor (VEGF) in MVs could stimulate angiogenesis. Therefore, a study was undertaken to confirm the presence of bone morphogenetic protein (BMP)-1 through-7, VEGF, and the noncollagenous matrix proteins, bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC), and osteonectin (ON) in isolated rat growth plate MVs. MVs were isolated from collagenase-digested rachitic rat tibial and femoral growth plates. The presence of BMP-1 through BMP-7, VEGF, BSP, ON, OPN, and OC was evaluated by Western blot, plus ELISA analyses for BMP-2 and-4 content. The alkaline phosphatase-raising ability of MV extracts on cultured rat growth plate chondrocytes was measured as a reflection of MV ability to promote chondroosseous differentiation. BMP-1 through-7, VEGF, BSP, ON, OPN, and OC were all detected by Western blot analyses. Chondrocytes treated with MV extracts showed a two-to threefold increase in alkaline phosphatase activity over control, indicating increased differentiation. Significant amounts of BMP-2 and BMP-4 were detected in MVs by ELISA. Combined, these data suggest that MVs could play an important morphogenetic role in growth plate and endochondral bone formation.

A novel three-dimensional stromal-based model for in vitro chemotherapy sensitivity testing of leukemia cells.

Abstract The disparate response of leukemia cells to chemotherapy in vivo, compared to in vitro, is partly related to the interaction of leukemic cells and the three-dimensional bone marrow stromal microenvironment. We investigated the effects of chemotherapy agents on leukemic cell lines co-cultured with human bone marrow mesenchymal stem cells (hu-BM-MSCs) in a three-dimensional model (3D). Comparison was made to leukemic cells treated in suspension, or grown on a hu-BM-MSC monolayer (2D conditions). We demonstrated that leukemic cells cultured in 3D were more resistant to drug-induced apoptosis compared to cells cultured in 2D or in suspension. We also demonstrated significant differences in leukemic cell response to chemotherapy using different leukemic cell lines cultured in 3D. We suggest that the differential responses to chemotherapy in 3D may be related to the expression of N-cadherin in the co-culture system. This unique model provides an opportunity to study leukemic cell responses to chemotherapy in 3D.

Effect of 25-hydroxyvitamin D3 and 1 α,25 dihydroxyvitamin D3 on differentiation and apoptosis of human osteosarcoma cell lines

Osteosarcoma (OS) is a malignant bone tumor predominantly affecting children and adolescents. OS has a 60% survival rate with current treatments; hence, there is a need to identify novel adjuncts to chemotherapeutic regimens. In this pilot study, we investigated the dose‐response to 1α,25‐dihdroxyvitamin D3 (1,α 25(OH)2D3) and 25‐hydroxyvitamin D3 (25(OH)D3) by human OS cell lines, SaOS‐2, and 143B. We hypothesized that 1,α 25(OH)2D3 and 25(OH)D3 would stimulate differentiation and induce apoptosis in OS cells in a dose‐dependent manner. Human OS cell lines, SaOS‐2, and 143B, were treated with 1,α 25(OH)2D3 or 25(OH)D3 or an ethanol control, respectively, at concentrations ranging from 1 to 1,000 nM. Ki67 (a marker of cellular proliferation) immunocytochemistry revealed no significant changes in the expression of Ki‐67 or MIB‐1 in 1α,25(OH)2D3 or 25(OH)D3 treated SaOS‐2 or 143B cells. Both control and 1α,25(OH)2D3 treated SaOS‐2 and 143B cells expressed vitamin D receptor (VDR). Markers of osteoblastic differentiation in 143B cells and SaOS‐2 cells were induced by both 25(OH)D3 and 1α,25(OH)2D, and evident by increases in alkaline phosphatase (ALP) activity, osteocalcin (OCN) mRNA expression, and mineralization of extra‐cellular matrix (ECM) by alizarin red staining. An increasing trend in apoptosis in response to 25(OH)D3, in both SaOS‐2 and 143B cells was detected by terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end labeling (TUNEL) staining. With 1α,25(OH)2D3 treatment, apoptosis was evident at higher concentrations only. These preliminary findings suggest that OS cells express VDR and respond to 25(OH)D3 and 1α,25(OH)2D3 by undergoing differentiation and apoptosis.

Expression of bone morphogenetic proteins and their receptors in the bone marrow megakaryocytes of GATA-1low mice: A possible role in osteosclerosis

The mechanism of osteosclerosis associated with myelofibrosis in megakaryocyte (MK)-related myeloproliferative disorders is largely unknown. However, growth factors released from the bone marrow cells, including from MKs, have been implicated in myelofibrosis, osteosclerosis, and angiogenesis. GATA-1 is a transcription factor required for normal MK development. GATA-1 deficiency in mice (GATA-1low) leads to increased megakaryocytic proliferation, followed by osteosclerosis and myelofibrosis. In this study we investigated the expression of bone morphogenetic proteins (BMPs) and BMP receptors and their possible role in the development of osteosclerosis in the MKs of 12-month-old GATA-1low mice by immunohistochemistry, cytomorphometry, and quantitative real-time PCR. Marrow MKs from both wild-type and GATA-1low mice showed moderate to intense staining for BMP-2, −4, and −6 and BMPR-IA and BMPR-II, whereas splenic MKs showed no BMP immunostaining. Presence of BMP protein in the bone marrow of GATA-1low mice was more than that seen in controls, owing to an increased number of MKs and osteoblasts. The osteosclerosis seen in GATA-1low mice appeared not to be due to a reduced number of functional osteoclasts because the number of tartrate-resistant acid phosphatase-positive osteoclasts was greater in GATA-1low mice than in controls. Our findings demonstrate the presence of significant amounts of BMP-2, −4, and −6 along with their receptors in bone marrow MKs of WT and GATA-1low mice. The increased levels of BMPs appear to be a result of increased numbers of MKs in GATA-1low mice and may, in part, account for the stimulation of osteoblastic activity and resulting osteosclerosis.

Extracellular Membrane Vesicles Derived from 143B Osteosarcoma Cells Contain Pro-Osteoclastogenic Cargo: A Novel Communication Mechanism in Osteosarcoma Bone Microenvironment

The bone microenvironment (BME) is the main hub of all skeletal related pathological events in osteosarcoma leading to tumor induced bone destruction, and decreasing overall bone quality and bone strength. The role of extra-cellular membrane vesicles (EMVs) as mediators of intercellular communication in modulating osteosarcoma-BME is unknown, and needs to be investigated. It is our hypothesis that osteosarcoma-EMVs contain pro-osteoclastogenic cargo which increases osteoclastic activity, and dysregulated bone remodeling in the osteosarcoma-BME. In this study, EMVs were isolated from the conditioned media of 143B and HOS human osteosarcoma cell cultures using differential ultracentrifugation. Nano-particle tracking analysis determined EMVs in the size range of 50-200 nm in diameter. The EMV yield from 143B cells was relatively higher compared to HOS cells. Transmission electron microscopy confirmed the ultrastructure of 143B-EMVs and detected multivesicular bodies. Biochemical characterization of 143B-EMVs detected the expression of bioactive pro-osteoclastic cargo including matrix metalloproteinases-1 and -13 (MMP-1, -13), transforming growth factor-β (TGF-β), CD-9, and receptor activator of nuclear factor kappa-β ligand (RANKL). Detection of a protein signature that is uniquely pro-osteoclastic in 143B-EMVs is a novel finding, and is significant as EMVs represent an interesting mechanism for potentially mediating bone destruction in the osteosarcoma-BME. This study further demonstrates that 143B cells actively mobilize calcium in the presence of ionomycin, and forskolin, and induce cytoskeleton rearrangements leading to vesicular biogenesis. In conclusion, this study demonstrates that 143B osteosarcoma cells generate EMVs mainly by mechanisms involving increased intracellular calcium or cAMP levels, and contain pro-osteoclastic cargo.

A simple and non-radioactive technique to study the effect of monophosphoesters on matrix vesicle-mediated calcification.

A simple and non-radioactive technique based on O-cresolpthalein complexone assay was developed to study in vitro non-radioactive calcium (40Ca) deposition by isolated matrix vesicles. Using this technique, the effect of various phosphoester substrates including ATP, AMP and β-GP on in vitro MV-calcification was studied. O-cresolpthalein complexone assay with non-radioactive calcium demonstrated that AMP or β-GP were more effective in promoting calcium deposition by isolated MVs than ATP. The application of this nonradioactive technique, which is highly sensitive and simple, would offer a useful alternative approach to the routinely used radiometric biomineralization assay which employs radioactive 45Ca.

Generating CK19-Positive Cells with Hair-Like Structures from Wharton's Jelly Mesenchymal Stromal Cells

Whartons jelly mesenchymal stromal cells (WJMSCs) are considered mesenchymal, multipotent, and capable of differentiating into cells of mesodermal origin. Ectodermal differentiation from mesenchymal cells has been recently reported. Herein, we show for the first time that we can generate cytokeratin 19-positive cells and hair-like structures from WJMSCs in vitro using 2 separate methodologies that utilize osteogenic media to induce WJMSCs to undergo osteogenic differentiation. In one method, WJMSCs were seeded on a matrix isolated from Whartons jelly following decellularization. In the other method, WJMSCs were cultured to form spheroids. Our findings demonstrate that WJMSCs may have the capacity for ectodermal differentiation.