Hemanth Akkiraju

Role of Chondrocytes in Cartilage Formation, Progression of Osteoarthritis and Cartilage Regeneration.

Articular cartilage (AC) covers the diarthrodial joints and is responsible for the mechanical distribution of loads across the joints. The majority of its structure and function is controlled by chondrocytes that regulate Extracellular Matrix (ECM) turnover and maintain tissue homeostasis. Imbalance in their function leads to degenerative diseases like Osteoarthritis (OA). OA is characterized by cartilage degradation, osteophyte formation and stiffening of joints. Cartilage degeneration is a consequence of chondrocyte hypertrophy along with the expression of proteolytic enzymes. Matrix Metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) are an example of these enzymes that degrade the ECM. Signaling cascades involved in limb patterning and cartilage repair play a role in OA progression. However, the regulation of these remains to be elucidated. Further the role of stem cells and mature chondrocytes in OA progression is unclear. The progress in cell based therapies that utilize Mesenchymal Stem Cell (MSC) infusion for cartilage repair may lead to new therapeutics in the long term. However, many questions are unanswered such as the efficacy of MSCs usage in therapy. This review focuses on the role of chondrocytes in cartilage formation and the progression of OA. Moreover, it summarizes possible alternative therapeutic approaches using MSC infusion for cartilage restoration.

Inhibition of CK2 binding to BMPRIa induces C2C12 differentiation into osteoblasts and adipocytes

BMP2 is a growth factor that regulates the cell fate of mesenchymal stem cells into osteoblast and adipocytes. However, the detailed signaling pathways and mechanism are unknown. We previously reported a new interaction of Casein kinase II (CK2) with the BMP receptor type-Ia (BMPRIa) and demonstrated using mimetic peptides CK2.1, CK2.2 and CK2.3 that the release of CK2 from BMPRIa activates Smad signaling and osteogenesis. Previously, we showed that mutation of these CK2 sites on BMPRIa (MCK2.1 (476S-A), MCK2.2 (324S-A) and MCK2.3 (214S-A)) induced osteogenesis. However, one mutant MCK2.1 induced osteogenesis similar to overexpression of wild type BMPRIa, suggesting that the effect of this mutant on mineralization was due to overexpression. In this paper we investigated the signaling pathways involved in the CK2-BMPRIa mediated osteogenesis and identified a new signaling pathway activating adipogenesis dependent on the BMPRIa and CK2 association. Further the mechanism for adipogenesis and osteogenesis is specific to the CK2 interaction site on BMPRIa. In detail our data show that overexpression of MCK2.2 induced osteogenesis was dependent on Caveolin-1 (Cav1) and the activation of the Smad and mTor pathways, while overexpression of MCK2.3 induced osteogenesis was independent of Caveolin-1 without activation of Smad pathway. However, MCK2.3 induced osteogenesis via the MEK pathway. The adipogenesis induced by the overexpression of MCK2.2 in C2C12 cells was dependent on the p38 and ERK pathways as well as Caveolin-1. These data suggest that signaling through BMPRIa used two different signaling pathways to induce osteogenesis dependent on CK2. Additionally the data supports a signaling pathway initiated in caveolae and one outside of caveolae to induce mineralization. Moreover, they reveal the signaling pathway of BMPRIa mediated adipogenesis.

Systemic injection of CK2.3, a novel peptide acting downstream of Bone Morphogenetic Protein receptor BMPRIa, leads to increased trabecular bone mass

Bone Morphogenetic Protein 2 (BMP2) regulates bone integrity by driving both osteogenesis and osteoclastogenesis. However, BMP2 as a therapeutic has significant drawbacks. We have designed a novel peptide CK2.3 that blocks the interaction of Casein Kinase 2 (CK2) with Bone Morphogenetic Protein Receptor type Ia (BMPRIa), thereby activating BMP signaling pathways in the absence of ligand. Here, we show that CK2.3 induced mineralization in primary osteoblast cultures isolated from calvaria and bone marrow stromal cells (BMSCs) of 8 week old mice. Further, systemic tail vein injections of CK2.3 in 8 week old mice resulted in increased bone mineral density (BMD) and mineral apposition rate (MAR). In situ immunohistochemistry of the femur found that CK2.3 injection induced phosphorylation of extracellular signal‐related kinase (ERK), but not Smad in osteocytes and osteoblasts, suggesting that CK2.3 signaling occurred through Smad independent pathway. Finally mice injected with CK2.3 exhibited decreased osteoclast differentiation and osteoclast activity. These data indicate that the novel mimetic peptide CK2.3 activated BMPRIa downstream signaling to enhance bone formation without the increase in osteoclast activity that accompanies BMP 2 stimulation.© 2014 Orthopaedic Research Society.

CK2.1, a novel peptide, induces articular cartilage formation in vivo

Bone morphogenetic protein 2 regulates chondrogenesis and cartilage formation. However, it also induces chondrocyte hypertrophy and cartilage matrix degradation. We recently designed three peptides CK2.1, CK2.2, and CK2.3 that activate the BMP signaling pathways by releasing casein kinase II (CK2) from distinct sites at the bone morphogenetic protein receptor type Ia (BMPRIa). Since BMP2 is a major regulator of chondrogenesis and the peptides activated BMP signaling in a similar way, we evaluated the effect of these peptides on chondrogenesis and cartilage formation. C3H10T1/2 cells were stimulated with CK2.1, CK2.2, and CK2.3 and evaluated for the chondrogenic and osteogenic potential. For chondrogenesis, Alcian blue staining was performed. Additionally, collagen types II and X expression was measured. For osteogenesis, osteocalcin and von Kossa staining were performed. From the three peptides, CK2.1 was the most promising peptide to induce chondrogenesis but not osteogenesis. To investigate the effect of CK2.1 on articular cartilage formation in vivo, we injected CK2.1 into the tail vein of mice. Injection of CK2.1 into the tail vein of mice led to increased articular cartilage formation but not BMD. In sharp contrast, injection of BMP2 led to increased BMD and expression of collagen type X, a marker of chondrocyte hypertrophy. MMP13 expression was unchanged. Our study demonstrates that CK2.1 drives chondrogenesis and cartilage formation without induction of chondrocyte hypertrophy. Peptide CK2.1 may, therefore, be a valuable therapeutic for cartilage degenerative diseases.

An Improved Immunostaining and Imaging Methodology to Determine Cell and Protein Distributions within the Bone Environment.

Bone is a dynamic tissue that undergoes multiple changes throughout its lifetime. Its maintenance requires a tight regulation between the cells embedded within the bone matrix, and an imbalance among these cells may lead to bone diseases such as osteoporosis. Identifying cell populations and their proteins within bone is necessary for understanding bone biology. Immunolabeling is one approach used to visualize proteins in tissues. Efficient immunolabeling of bone samples often requires decalcification, which may lead to changes in the structural morphology of the bone. Recently, methyl-methacrylate embedding of non-decalcified tissue followed by heat-induced antigen retrieval has been used to process bone sections for immunolabeling. However, this technique is applicable for bone slices below 50-µm thickness while fixed on slides. Additionally, enhancing epitope exposure for immunolabeling is still a challenge. Moreover, imaging bone cells within the bone environment using standard confocal microscopy is difficult. Here we demonstrate for the first time an improved methodology for immunolabeling non-decalcified bone using a testicular hyaluronidase enzyme-based antigen retrieval technique followed by two-photon fluorescence laser microscopy (TPLM) imaging. This procedure allowed us to image key intracellular proteins in bone cells while preserving the structural morphology of the cells and the bone.

Targeting of Calcitriol to Inflammatory Breast Cancer Tumors andMetastasis In Vitro and In Vivo

Inflammatory Breast Cancer (IBC) is arguably the most severe form of breast cancer with a very high mortality rate. IBC is distinct from other forms of breast cancer expressing unique cell surface markers such as hypoglycosylated MUC1. However, treatment options for IBC are limited. Calcitriol is a potential treatment for IBC due to its potential role as a therapeutic in other forms of cancer. Our previous research demonstrated that calcitriol inhibits the metastatic ability of the SUM149 IBC cell line. However, high concentration of calcitriol would be required for treatment. This may result I serious side effects such as hypercalcemia. Targeting calcitriol directly to the tumor site would allow for treatment without toxic levels of calcitriol. Here we developed SM3MUC1 antibody conjugated calcitriol bound QDs as a novel nanoparticles probe. We demonstrate that these particles can be used to target to MUC1 over-expressing IBC cells in vitro and in vivo. Therefore these particles can be used to determine the localization of IBC emboli in vivo and maybe used as a potential vehicle to deliver high doses of calcitriol to the IBC tumors and metastasis.

Current Challenges in Bone Biology

The bone is a very important organ that supports is one of the many bodily functions. It has very diverse functions from the general support of the human body to energy regulation and balance [1]. Bones are formed during the development either through intramembranous (flat bones) ossification or endochondral ossification (long bones) [2–4]. The human body is composed of over 270 bones at birth and fuse to become 206 in totals at adulthood that all hold crucial functions. Bones consisting of mineralized bone tissue also consists of bone marrow, nerves and blood vessels and the communication between cells in the tissues is tightly regulated by the bone environment. Bone is an active tissue that is maintained by bone cells such as osteoblasts that form bone and osteoclasts that resorb bone [5]. Additionally, within the collagen and mineral matrix osteocytes are also embedded and respond to the bone environment [6,7]. The balance between these cells is necessary to maintain bone function. Bone research is considerably a challenging field due to the intricately dense structural composition of the bone morphology. While other tissues can be easily processed and prepared for experiments, working with bone is difficult [8,9]. Due to its composition of collagen fibers and minerals, bone creates a very dense structure, in which the bone cells are embedded [10]. Therefore, studying intracellular dynamics of the bone cells embedded within the mineralized tissue has proven to be challenging a task. Imaging cells at subcellular level within the bone environment is very challenging. Conventional intracellular studies are performed on decalcified thin tissue slices embedded in paraffin [9]. However, this kind of bone sample preparation can lead to significant changes in bones biochemical properties of antigenicity and to its mineral structure [9]. Alternatively, non-decalcified bone samples can be processed in resin based polymers and be labeled fluorescently for target proteins [8,11,12]. However, current methods are tedious and very limited. Most approaches used to image cells within the bone such as MRI, Micro-CT or Ultrasound can image bone structure and recently cells, however these techniques are limited by their low resolution at the cellular level [13]. Tissues embedded within the bone itself such as the bone marrow niche and blood vessels are easier to analyze. For example real time imaging of the bone marrow niche within bone was recently achieved [14,15]. Similarly, fluorescent imaging of cells within the bone marrow niche was also achieved [16]. However, determining the localization of cell types and protein expression dynamics of single cells within the bone is still very difficult. Recent advancements in imaging techniques allows for the identification of osteocytes embedded in the bone matrix [17]. However, more research is needed to identify intracellular protein activities of the cell bodies embedded within mineralized matrix. Alternatively, researchers study cell dynamics in ex vivo models. Several ex vivo models of bone are developed to study cellular dynamics of bone [18–21]. These novels ex vivo bone cultures are proposed for studying inflammatory responses, cancer metastasis, and also Zetos bone bioreactor used to study bone growth utilizing mechanosensitive loading are a few good examples [19,22,23]. These ex vivo models can overcome many ethical and clinical issues that are otherwise not permissive for animal or human trials. Such model systems also allow for the imaging of bone cells more feasible [24]. One model for example uses trabecular bone samples and replaces the cells in the 3D architecture with live cells. This allows for a controlled environment within the ex vivo model structure to study bone cell function [25]. Other researchers try to recreate specific bone environments for cells by using hydrogels or porous microspheres to support 3D growth of cells [26,27]. As these data show the cells in a 3D environment show often completely different cellular dynamics as compared to their 2D cultures [28]. Although mimicking the tissue environment through ex vivo model systems makes a significant breakthrough in testing cellular responses but it is still hard to replicate the exact environmental processes. To better understand bone function we desperately need the development of new protocols and methods to drive bone research. This is especially important to address the cause of bone diseases and their possible treatment options. Bone diseases such as osteoporosis tremendously impact on the quality of life of individuals. Musculoskeletal diseases affect one out of every two people in the United States age 18 and over, and nearly three out of four age 65 and over [29]. However, in order to develop treatments one needs to understand the basic cellular mechanisms first.