A comparison of human mesenchymal stem cell osteogenesis in poly(ethylene glycol) hydrogels as a function of MMP-sensitive crosslinker and crosslink density in chemically defined medium

This study investigated osteogenesis of human mesenchymal stem cells encapsulated in matrix-metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG) hydrogels in chemically defined medium (10 ng/ml bone morphogenic factor-2). Thiol-norbornene photoclick hydrogels were formed with CRGDS and crosslinkers of PEG dithiol (nondegradable), CVPLS-LYSGC (P1) or CRGRIGF-LRTDC (P2; dash indicates cleavage site) at two crosslink densities. Exogenous MMP-2 degraded P1 and P2 hydrogels similarly. MMP-14 degraded P1 hydrogels more rapidly than P2 hydrogels. Cell spreading was greatest in P1 low crosslinked hydrogels and to a lesser degree in P2 low crosslinked hydrogels, but not evident in nondegradable and high crosslinked MMP-sensitive hydrogels. Early osteogenesis (Alkaline phosphatase [ALP] activity) was accelerated in hydrogels that facilitated cell spreading. Contrarily, late osteogenesis (mineralization) was independent of cell spreading. Mineralized matrix was present in P1 hydrogels, but only present in P2 high crosslinked hydrogels and not yet present in nondegradable hydrogels. Overall, the low crosslinked P1 hydrogels exhibited an accelerated early and late osteogenesis with the highest ALP activity (Day 7), greatest calcium content (Day 14), and greatest collagen content (Day 28), concomitant with increased compressive modulus over time. Collectively, this study demonstrates that in chemically defined medium, hydrogel degradability is critical to accelerating early osteogenesis, but other factors are important in late osteogenesis.     

Gaseous nitric oxide exhibits minimal effect on skin fibroblast extracellular matrix gene expression and immune cell viability

NO (nitric oxide) molecule is produced by various mammalian cell types and plays a significant role in inflammation, infection and wound healing processes. Recently, gNO (gaseous nitric oxide) therapy has been utilized for its potential clinical application as an antimicrobial agent, with special focus on skin infection. In a previous study, we demonstrated that 200 ppm gNO, 8 h/day for three consecutive days significantly reduced the number of bacteria in dermal wounds without compromising the viability and function of skin cells. To increase the feasibility and ease of its clinical use, we propose that different doses of gNO (5 to 10 K ppm) for 8 h and as short as 10 min be used, respectively. To achieve this, we set up in vitro experiments and asked whether (i) different doses of gNO have any toxic effect on immune cells and (ii) gNO has any modulating effect on key ECM (extracellular matrix) components in fibroblasts. To further investigate the effect of gNO, expression of more than 100 key ECM genes have been examined using gene array in human fibroblasts. As immune cells play an important role in wound healing, the effect of gNO on proliferation and viability of human and mouse lymphocytes was also examined. The findings showed that, the 5, 25, 75 and 200 ppm of gNO for 8 h slightly increased the expression of Col 5A3 (collagen type V alpha 3), and gNO at 5 ppm decreased the expression of MMP-1 (matrix metalloproteinase 1), while exposure of fibroblast to 10 K ppm of gNO for 10 min does not show any significant changes in ECM genes. Exposure to gNO resulted in inhibition of lymphocyte proliferation without affecting the cell viability. Taken together, our findings show that skin could be treated with gNO without compromising the role of ECM and immune cells in low concentrations with long time exposure or high concentrations for a shorter exposure time.     

Decellularized breast matrix as bioactive microenvironment for in vitro three-dimensional cancer culture

Breast cancer (BC) is a leading cause of cancer-related death in women with unsatisfactory survival rates. Advances in the understanding of the genetic basis of BC provide the opportunity to develop gene-based medicines capable of treating metastatic diseases. Here, we first demonstrated efficient tissue engineering approaches applied to normal breast and BC extracellular matrix (ECM) starting from decellularized human biopsies to generate a three-dimensional (3D) bioactive model with the sodium lauryl ether sulfate solution. The decellularized tissues maximized the genetic component removal from tissues and minimally injured ECM structures and native compositions by histology and ECM compositions analyses. Importantly, we proved that the 3D ECM retained tissues biological properties. We demonstrated that after 30 days of recellularization with MCF-7 cell (human breast adenocarcinoma cell line), the 3D cancer ECM induced an overexpression of epithelial–mesenchymal transition (EMT) and cancer proliferation. Meanwhile, normal ECM from the breast inhibited EMT and cell growth with the inducement of apoptosis. Given the biological activity preserved in the ECM after decellularization, we believe these approaches are powerful tools for future preclinical research for BC and breast development.     

Cloning,Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells

A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1–240) and truncated (residues 19–240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.     

Extracellular matrix protein laminin enhances mesenchymal stem cell (MSC) paracrine function through αvβ3/CD61 integrin to reduce cardiomyocyte apoptosis

Myocardial ischaemia (MI) results in extensive cardiomyocyte death and reactive oxygen species (ROS)‐induced damage in an organ with little or no regenerative capacity. Although the use of adult bone marrow mesenchymal stem cells (BMMSCs) has been proposed as a treatment option, the high cell numbers required for clinical use are difficult to achieve with this source of MSCs, and animal studies have produced inconsistent data. We recently demonstrated in small and large animal models of acute MI that the application of human term placenta‐derived multipotent cells (PDMCs), a foetal‐stage MSC, resulted in reversal of cardiac injury with therapeutic efficacy. However, the mechanisms involved are unclear, making it difficult to strategize for therapeutic improvements. We found that PDMCs significantly reduced cardiomyocyte apoptosis and ROS production through the paracrine factors GRO‐α, HGF and IL‐8. Moreover, culturing PDMCs on plates coated with laminin, an extracellular matrix (ECM) protein, resulted in significantly enhanced secretion of all three paracrine factors, which further reduced cardiomyocyte apoptosis. The enhancement of PDMC paracrine function by laminin was mediated through αvβ3 integrin, with involvement of the signalling pathways of JNK, for GRO‐α and IL‐8 secretion, and PI3K/AKT, for HGF secretion. Our results demonstrated the utility of PDMC therapy to reduce cardiomyocyte apoptosis through modulation of ECM proteins in in vitro culture systems as a strategy to enhance the therapeutic functions of stem cells.     

In vitro lung cancer multicellular tumor spheroid formation using a microfluidic device

The purpose of this study was to demonstrate self-organizing in vitro multicellular tumor spheroid (MCTS) formation in a microfluidic system and to observe the behavior of MCTSs under controlled microenvironment. The employed microfluidic system was designed for simple and effective formation of MCTSs by generating nutrient and oxygen gradients. The MCTSs were composed of cancer cells, vascular endothelial cells, and type I collagen matrix to mimic the in vivo tumor microenvironment (TME). Cell culture medium was perfused to the microfluidic device loaded with MCTSs by a passive fluidic pump at a constant flow rate. The dose response to an MMPs inhibitor was investigated to demonstrate the effects of biochemical substances. The result of long-term stability of MCTSs revealed that continuous perfusion of cell culture medium is one of the major factors for the successful MCTS formation. A continuous flow of cell culture medium in the in vitro TME greatly affected both the proliferation of cancer cells in the micro-wells and the sustainability of the endothelial cell-layer integrity in the lumen of microfluidic channels. Addition of MMP inhibitor to the cell culture medium improved the stability of the collagen matrix by preventing the detachment and shrinkage of the collagen matrix surrounding the MCTSs. In summary, the present constant flow assisted microfluidic system is highly advantageous for long-term observation of the MCTS generation, tumorous tissue formation process and drug responses. MCTS formation in a microfluidic system may serve as a potent tool for studying drug screening, tumorigenesis and metastasis.     

Understanding the role of chondrocytes in osteoarthritis: utilizing proteomics

Introduction: Proteomic analyses have been acknowledged to carry a significant prospective in elucidating the pathogenesis of several diseases, including osteoarthritis (OA). But it has not been an easy road: major technical issues, mainly derived from the complex and rigid nature of the cartilage tissue, had to be faced; an obstacle that led to the development of different approaches.

Areas covered: In this review, we categorized the proteomic studies undertaken (proteomic analyses of the cartilage, cartilage explants, cultured chondrocytes, and chondrocytes’ secretome) as part of the different strategies developed in order to overcome tissue and disease-specific challenges. Essentially these approaches aimed at identifying differences in the proteome of healthy vs diseased tissue. Our aim was to point out the novel players that have emerged from these analyses and highlight the associated mechanism(s) suggested to play a role in the pathogenesis of OA.

Expert commentary: The identified factors indicate the implication of age-associated mechanisms, such as metabolic deregulation, inflammation, and redox imbalance, in OA onset and/or progression. Taken together these results outline the causal network of the disease and place chondrocytes’ senescence at the center of the emerging aetiopathological atlas.     

Extracellular matrix (ECM) stiffness and degradation as cancer drivers

Alteration in the density and composition of extracellular matrix (ECM) occurs in tumors. The alterations toward both stiffness and degradation are contributed to tumor growth and progression. Cancer-associated fibroblasts (CAFs) are the main contributors to ECM stiffness and degradation. The cells interact with almost all cells within the tumor microenvironment (TME) that could enable them to modulate ECM components for tumorigenic purposes. Cross-talks between CAFs with cancer cells and macrophage type 2 (M2) cells are pivotal for ECM stiffness and degradation. CAFs induce hypoxia within the TME, which is one of the key inducers of both stiffness and degradation. Cancer cell modulatory roles in integrin receptors are key for adjusting ECM constituents to either fates. Cancer cell proliferation, migration, and invasion as well as angiogenesis are consequences of ECM stiffness and degradation. ECM stiffness in a transforming growth factor-β (TGF-β) related pathway could make a bridge in the basement membrane, and ECM degradation in a matrix metalloproteinase (MMP)-related pathway could make a path in the TME, both of which contribute to cancer cell invasion. ECM stiffness is also obstructive for drug penetration to the tumor site. Therefore, it would be a promising strategy to make a homeostasis in ECM for easy penetration of chemotherapeutic drugs and increasing the efficacy of antitumor approaches. MMP and TGF-β inhibitors, CAF and M2 reprogramming toward their normal counterparts, reduction of TME hypoxia and hampering integrin signaling are among the promising approaches for the modulation of ECM in favor of tumor regression.     

Intestine-on-chip device increases ECM remodeling inducing faster epithelial cell differentiation

An intestine-on-chip has been developed to study intestinal physiology and pathophysiology as well as intestinal transport absorption and toxicity studies in a controlled and human similar environment. Here, we report that dynamic culture of an intestine-on-chip enhances extracellular matrix (ECM) remodeling of the stroma, basement membrane production and speeds up epithelial differentiation. We developed a three-dimensional human intestinal stromal equivalent composed of human intestinal subepithelial myofibroblasts embedded in their own ECM. Then, we cultured human colon carcinoma-derived cells in both static and dynamic conditions in the opportunely designed microfluidic system until the formation of a well-oriented epithelium. This low cost and handy microfluidic device allows to qualitatively and quantitatively detect epithelial polarization and mucus production as well as monitor barrier function and ECM remodeling after nutraceutical treatment.     

Spatial and temporal regulation of collagenases—3,—4,and stromelysin —3 implicates distinct functions in apoptosis and tissue remodeling during frog metamorphosis

Matrix metalloproteinases (MMPs) are a family of extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix(ECM).They have been implicated to play important roles in a number of developmental and pathological processes,such as tumor metastasis and inflammation.Relatively few studies have been carried out to investigate the function of MMPs during postembryonic organ-development.Using Xenopus laevis development as a model system,we demonstrate here that three MMPs,stromelysin-3(ST3),collagenases-3(Col3),and Col4,have distinct spatial and temporal expression profiles during metamorphosis as the tadpole transforms into a frog.In situ hybridizations reveal a tight,but distinct,association of individual MMPs with tissue remodeling in the tail and intestine during metamorphosis.In particular,ST3 expression is strongly correlated with apoptosis in both organs as demonstrated by analyses of serial sections with in situ hybridization for ST3 mRNA and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling)for apoptosis,respectively.On the other hand,Col3 and Col4 are present in regions where extensive connective tissue remodeling take place.These results indicate that ST3 is likely to play a role in ECM-remodeling that facilitate apoptotic tissue remodeling or resorption while Col3 and Col4 appear to participate in connective tissue degradation during development.     

Induction of SC1 mRNA Encoding a Brain Extracellular Matrix Glycoprotein Related to SPARC following Lesioning of the Adult Rat Forebrain

SC1 is an extracellular matrix (ECM) glycoprotein related to SPARC which exhibits anti-adhesive properties. ECM molecules are thought to play important roles in influencing cell shape, proliferation and migration during neurogenesis. Following localized injury to the adult rat forebrain, a biphasic induction of SC1 mRNA was apparent, namely a rapid, transient induction at 1 day post-lesion in cortical neurons which border the lesion site followed by a more prolonged induction in astrocytes which are proximal to the wound site. A similar SC1 induction pattern was observed in the hippocampus in response to the injury. SPARC mRNA exhibits a divergent pattern of induction because it is induced in mature blood vessels close to the lesion and in blood vessels which develop following the trauma. Thus mRNAs encoding the related ECM glycoproteins SC1 and SPARC are induced in different cell populations in the adult forebrain during the neural response to localized injury.     

The effect of TGF-beta1 and beta-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures

Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell-matrix interaction, cytoskeletal organization and integrin-mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin-like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF-beta1 and beta-estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF-beta1 and beta-estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10ng/ml of TGF-beta1, 10nM of beta-estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF-beta1 treated cultures. This increase was also noted when TGF-beta1 and beta-estradiol were both used as culture supplements. However, beta-estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF-beta1 when compared with cultures treated with combined factors. From this, we conclude that although TGF-beta1 appears to stimulate chondrocyte ECM synthesis, beta-estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, beta-estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF-beta1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.     

Cellular remodelling of individual collagen fibrils visualized by time-lapse AFM

The extracellular matrix in tissues such as bone, tendon and cornea contains ordered, parallel arrays of collagen type I fibrils. Cells embedded in these matrices frequently co-align with the collagen fibrils, suggesting that ordered fibrils provide structural or signalling cues for cell polarization. To study mechanisms of matrix-induced cell alignment, we used nanoscopically defined two-dimensional matrices assembled of highly aligned collagen type I fibrils. On these matrices, different cell lines expressing integrin alpha(2)beta(1) polarized strongly in the fibril direction. In contrast, alpha(2)beta(1)-deficient cells adhered but polarized less well, suggesting a role of integrin alpha(2)beta(1) in the alignment process. Time-lapse atomic force microscopy (AFM) demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization was prevented. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix, leading to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction.     

The fibrous system in the extracellular matrix of hydromedusae

The ultrastructure and the histochemistry of the fibrous system in the mesogloeal extracellular matrix (ECM) of two hydromedusae (Polyorchis penicillatus and Aglanlha digitale) has been examined. There is a fundamental difference in the architecture of the fibrous system between the two species. In Polyorchis, 60-150 A thick, striated fibrils with periodicities of 60-65 A form a three-dimensional network which fills in the entire ECM of outer and inner mesogloea. In the outer mesogloea vertical fibres (up to 1.8 mum in diameter) penetrate the threedimensional network and branch near the exumbrellar and subumbrellar side. These branches impinge on a dense matrix covering the exumbrellar and subumbrellar surface. In Aglantha the branches of thick vertical fibres anchor at the subumbrellar side in a dense plexus (0.2-0.3 mum in thickness) which consists of two types of fibrils (35-40 and 80-100 nm in diameter). Towards the exumbrellar side the vertical fibres branch and intermingle with a meshwork of non-striated fibrils with uniform diameter (35-40 nm). These fibrils form a laminated structure (about 1 mum in thickness) so that fibrils of each layer course in the same direction but fibrils of adjacent layers run perpendicularly to each other. The banded pattern with periodicities of 600-640 A observed in the electron microscope and by histochemical methods confirm the thick vertical fibres and their branches to be a collagen. There is also strong evidence that the laminated structure in Aglantha represents layers of collagen fibrils.     

整合素在细胞响应机械应力中的作用

机械应力在细胞生长、分化和基因表达等生理学过程和某些病理学过程中起了重要的作用.细胞粘附分子——整合素是机械信号转导中重要的跨膜分子.细胞通过整合素与胞外基质蛋白、细胞骨架蛋白以及聚焦粘附激酶等的反应,将感应的力信号转化为化学信号,从而调节细胞的生理机能,其中整合素与胞外基质蛋白之间的动态和特异性反应在细胞的机械信号转导过程中起了功能性作用.     

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(ω). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.     

Cellular localization of integrin isoforms in phenylephrine-induced hypertrophic cardiac myocytes

Cardiac hypertrophy is characterized by remodeling of the extracellular matrix (ECM). Integrins are cell-surface molecules that link the ECM to the cellular cytoskeleton where they play roles as signaling molecules and transducers of mechanical force. To clarify the possible roles of integrins in cardiac myocyte hypertrophy, we investigated the cellular localization and expression of ECM proteins and integrins in both normal cardiac myocytes and phenylephrine-induced hypertrophic myocytes. Addition of phenylephrine (PE) to cultured neonatal cardiac myocytes induced sarcomeric organization, increase in cell size, and synthesis of the hypertrophic marker, atrial natriuretic factor (ANF). In particular, fibronectin and collagen underwent dramatic localization changes during PE-induced cardiac hypertrophy. Significant changes were noted in the cellular localization of the respective collagen and fibronectin receptors, integrin alpha1 and alpha5, from diffuse to a sarcomeric banding pattern. Expression levels of integrins were also increased during hypertrophy. Treatment with okadaic acid (OA), an inhibitor of protein phosphatase 2A (PP2A), resulted in inhibition of hypertrophic response. These results suggest that dephosphorylation of integrin beta1 may be important in the induction of cardiac hypertrophy.     

Regulation of collagen fibrillogenesis by cell-surface expression of kinase dead DDR2

The assembly of collagen fibers, the major component of the extracellular matrix (ECM), governs a variety of physiological processes. Collagen fibrillogenesis is a tightly controlled process in which several factors, including collagen binding proteins, have a crucial role. Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that bind to and are phosphorylated upon collagen binding. The phosphorylation of DDRs is known to activate matrix metalloproteases, which in turn cleave the ECM. In our earlier studies, we established a novel mechanism of collagen regulation by DDRs; that is, the extracellular domain (ECD) of DDR2, when used as a purified, soluble protein, inhibits collagen fibrillogenesis in-vitro. To extend this novel observation, the current study investigates how the DDR2-ECD, when expressed as a membrane-anchored, cell-surface protein, affects collagen fibrillogenesis by cells. We generated a mouse osteoblast cell line that stably expresses a kinase-deficient form of DDR2, termed DDR2/-KD, on its cell surface. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays demonstrated that the expression of DDR2/-KD reduced the rate and abundance of collagen deposition and induced significant morphological changes in the resulting fibers. Taken together, our observations extend the functional roles that DDR2 and possibly other membrane-anchored, collagen-binding proteins can play in the regulation of cell adhesion, migration, proliferation and in the remodeling of the extracellular matrix.     

Human peripheral blood mononuclear cells: A review of recent proteomic applications

Human peripheral blood mononuclear cells (PBMCs) represent a sentinel blood sample which reacts to different pathophysiological stimuli in the form of immunological responses/immunophenotypic changes. The study of molecular content of PBMCs can provide better understanding of immune processes giving the possibility of monitoring the health conditions of the host organism. Proteomic analysis of PBMCs can achieve mentioned goal as important immune-related biomarkers are easily accessible for analysis. PBMCs have been gaining attention in different research areas including preclinical or clinical investigations. In this review, recent applications of proteomic analysis of PBMCs are described and discussed. Approaches are divided based on different proteomic workflows such as in-gel, in-solution and on-filter modes. The effect of various diseases such as autoimmune, cancer, neurodegenerative, viral, metabolic, and various immune stimulations such as radiation, vaccine, corticosteroids over PBMCs proteome, are described with emphasis on promising protein biomarker candidates.