Exosomes as a storehouse of tissue remodeling proteases and mediators of cancer progression.

Rapidly increasing scientific reports of exosomes and their biological effects have improved our understanding of their cellular sources and their cell-to-cell communication. These nano-sized vesicles act as potent carriers of regulatory bio-macromolecules and can induce regulatory functions by delivering them from its source to recipient cells. The details of their communication network are less understood. Recent studies have shown that apart from delivering its cargo to the cells, it can directly act on extracellular matrix (ECM) proteins and growth factors and can induce various remodeling events. More importantly, exosomes carry many surface-bound proteases, which can cleave different ECM proteins and carbohydrates and can shed cell surface receptors. These local extracellular events can modulate signaling cascades, which consequently influences the whole tissue and organ. This review aims to highlight the critical roles of exosomal proteases and their mechanistic insights within the cellular and extracellular environment.

Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights.

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.

Matrix metalloproteinases: The sculptors of chronic cutaneous wounds.

Cutaneous wound healing is a complex mechanism with multiple processes orchestrating harmoniously for structural and functional restoration of the damaged tissue. Chronic non-healing wounds plagued with infection create a major healthcare burden and is one of the most frustrating clinical problems. Chronic wounds are manifested by prolonged inflammation, defective re-epithelialization and haphazard remodeling. Matrix metalloproteinases (MMPs) are zinc dependent enzymes that play cardinal functions in wound healing. Understanding the pathological events mediated by MMPs during wound healing may pave way in identifying novel drug targets for chronic wounds. Here, we discuss the functions and skewed regulation of different MMPs during infection and chronic tissue repair. This review also points out the potential of MMPs and their inhibitors as therapeutic agents in treating chronic wounds during distinct phases of the wound healing. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Fabrication of electrospun zein nanofibers for the sustained delivery of siRNA.

In this study, zein nanofibers based siRNA delivery system has been attempted for the first time. Here, the amphiphilic property of zein and the size advantage of nanofibers have been brought together in developing an ideal delivery system for siRNA. The morphological analysis of the GAPDH-siRNA loaded zein nanofibers revealed the proper encapsulation of the siRNA in the polymeric matrix. The loading efficiency of this delivery system was found to be 58.57±2.4% (w/w). The agarose gel analysis revealed that the zein nanofibers preserved the integrity of siRNA for a longer period even at the room temperature. The in vitro release studies not only depicted the sustaining potential of the zein nanofibers but also ensured the release of sufficient quantity of siRNA required to induce the gene silencing effect. The amphiphilic property of zein supported the cell attachment and thereby facilitated the transfection of siRNA into the cells. qRT-PCR analysis confirmed the potential of the developed system in inducing the desired gene silencing effect. Thus, electrospun zein nanofibers have been successfully employed for the delivery of siRNA which has a great therapeutic potential.

Expression and integrity of dermatopontin in chronic cutaneous wounds: a crucial factor in impaired wound healing.

Chronic cutaneous wound (CCW) is a major health care burden wherein the healing process is slow or rather static resulting in anatomical and functional restriction of the damaged tissue. Dysregulated expression and degradation of matrix proteins, growth factors and cytokines contribute to the disrupted and uncoordinated healing process of CCW. Therefore, therapeutic approaches for effective management of CCW should be focused towards identifying and manipulating the molecular defects, such as reduced bioavailability of the pro-healing molecules and elevated activity of proteases. This study essentially deals with assessing the expression and integrity of an extracellular matrix protein, Dermatopontin (DPT), in CCW using real-time quantitative reverse transcriptase PCR and immunological techniques. The results indicate that, despite DPT's high mRNA expression, the protein levels are markedly reduced in both CCW tissue and its exudate. To elucidate the cause for this contradiction in mRNA and protein levels, the stability of DPT is analyzed in the presence of wound exudates and various proteases that are naturally elevated in CCW. DPT was observed to be degraded at higher rates when incubated with certain recombinant proteases or chronic wound exudate. In conclusion, the susceptibility of DPT protein to specific proteases present at high levels in the wound milieu resulted in the degradation of DPT, thus leading to impaired healing response in CCW.

A naphthalimide based PET probe with Fe3+ selective detection ability: theoretical and experimental study.

A naphthalimide based fluorescent probe '1' that operates based on photoinduced electron transfer phenomenon is synthesized and its chemosensory application is explored. Among various metal ions, 1 selectively detects Fe(3+) with a detection limit of 3.0 × 10(-8) M. 1 is stable at physiological pH, nontoxic under experimental conditions and suitable for the detection of Fe(3+) ions present in aqueous samples and live cells.

Probe-free optical chromatin deformation and measurement of differential mechanical properties in the nucleus.

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C/µm) can lead to substantial intra-nuclear chromatin displacements (>1 µm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high resistance and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Longitudinal in vivo imaging of perineuronal nets.

Significance: Perineuronal nets (PNNs) are extracellular matrix structures implicated in learning, memory, information processing, synaptic plasticity, and neuroprotection. However, our understanding of mechanisms governing the evidently important contribution of PNNs to central nervous system function is lacking. A primary cause for this gap of knowledge is the absence of direct experimental tools to study their role in vivo. Aim: We introduce a robust approach for quantitative longitudinal imaging of PNNs in brains of awake mice at subcellular resolution. Approach: We label PNNs in vivo with commercially available compounds and monitor their dynamics with two-photon imaging. Results: Using our approach, we show that it is possible to longitudinally follow the same PNNs in vivo while monitoring degradation and reconstitution of PNNs. We demonstrate the compatibility of our method to simultaneously monitor neuronal calcium dynamics in vivo and compare the activity of neurons with and without PNNs. Conclusion: Our approach is tailored for studying the intricate role of PNNs in vivo, while paving the road for elucidating their role in different neuropathological conditions.

Multi-well Fibrillation Assay as a Tool for Analyzing Crosslinking and Stabilization of Collagen.

Collagen is the most widely used substratum in cell culture and biomaterials applications. In this chapter, we describe a simple procedure to isolate collagen, which can be employed to a wide range of tissue sources, and subsequently use it to study the collagen crosslinking and stabilization abilities of various compounds. The protocol is designed for a multi-well format assay and thus can be used for simultaneous assessment of multiple number of compounds and can be easily adapted to a high-throughput screening setup.

Dermatopontin augments angiogenesis and modulates the expression of transforming growth factor beta 1 and integrin alpha 3 beta 1 in endothelial cells.

Dermatopontin (DPT) is a matricellular protein with cardinal roles in cutaneous wound healing. The protein is also reported to be altered in various anomalies including cancer. The present study is aimed to unravel the role of DPT in angiogenesis which is imperative in many physiological and pathological processes. DPT's capabilities on promoting angiogenesis were assessed using various in vitro and ex vivo systems. The results indicated that DPT enhances cell motility and induces lamellipodia formation in endothelial cells. Additionally, we noticed that DPT stimulates tube formation in endothelial cells when plated on a matrigel substrate. However, it was observed that DPT had no effect on the proliferation of endothelial cells even at higher concentrations and prolonged treatment periods. Additional experiments on CAM and aortic arch assays apparently depicted that DPT promotes neovascularisation and tube sprouting, thus unraveling its prominent role in angiogenesis. Further, PCR analysis revealed that endothelial cells are devoid of DPT expression, but when exogenously supplied, modulates the expression of transforming growth factor ß1 and integrin α3ß1 which are reported to have crucial roles in endothelial cell behaviour during angiogenesis. In conclusion, DPT possess vital pro-angiogenic properties and thus retains promising therapeutic values in managing chronic wounds and cancer.

Recent advancements in nanotechnological strategies in selection, design and delivery of biomolecules for skin regeneration.

Skin is a very complex organ and hence designing a bioengineered skin model replicating the essential physiological characteristics for replacing the diseased or damaged parts has been a challenging goal for many. Newer technologies for satisfying most of the criteria are being attempted with the copious efforts of biologists, engineers, physiologists, using multitude of features in combination. Amongst them nanotechnology based biomaterials have gained prominence owing to the enhanced pharmacokinetics, bio-distribution profile, extended half-life and reduced side effects. Designing a matrix that can be assimilated into the body during the regeneration and delivering the essential pharmacological agents in a temporal and spatially specific manner is a tremendous goal. This review essentially deals with the various approaches for designing a multidisciplinary translational smart matrix for addressing the various skin related ailments.

A novel FRET 'off-on' fluorescent probe for the selective detection of Fe³âº, Al³âº and Cr³âº ions: its ultrafast energy transfer kinetics and application in live cell imaging.

A rhodamine-naphthalimide dyad probe, 1, that selectively responds to the addition of trivalent metal ions (Fe(3+) or Al(3+) or Cr(3+)) via ultrafast Förster resonance energy transfer (FRET) from naphthalimide to rhodamine is designed and synthesized. 1 is highly selective to the trivalent metal ions and the presence of other monovalent or divalent metal ions do not affect its detection ability. The probe is highly sensitive and it can respond to the presence of trivalent metal ions even at sub-micromolar levels. 1 is stable over a broad range of pH, non-toxic under experimental conditions and suitable to the fluorescence bio-imaging of live cells exposed to trivalent metal ions. The trivalent metal ion induced ultrafast energy transfer kinetics of 1 is explored using time resolved fluorescence experiments.

Role of dermatopontin in re-epithelialization: implications on keratinocyte migration and proliferation.

Re-epithelialization is a key event in wound healing and any impairment in that process is associated with various pathological conditions. Epidermal keratinocyte migration and proliferation during re-epithelialization is largely regulated by the cytokines and growth factors from the provisional matrix and dermis. Extracellular matrix consists of numerous growth factors which mediate cell migration via cell membrane receptors. Dermatopontin (DPT), a non-collagenous matrix protein highly expressed in dermis is known for its striking ability to promote cell adhesion. DPT also enhances the biological activity of transforming growth factor beta 1 which plays a central role in the process of wound healing. This study was designed to envisage the role of DPT in keratinocyte migration and proliferation along with its mRNA and protein expression pattern in epidermis. The results showed that DPT promotes keratinocyte migration in a dose dependant fashion but fail to induce proliferation. Further, PCR and immunodetection studies revealed that the mRNA and protein expression of DPT is considerably negligible in the epidermis in contrast to the dermis. To conclude, DPT has a profound role in wound healing specifically during re-epithelialization by promoting keratinocyte migration via paracrine action from the underlying dermis.

Fabrication of highly aligned fibrous scaffolds for tissue regeneration by centrifugal spinning technology.

Centrifugal spinning (C-Spin) is an emerging technology which uses centrifugal force to produce ultrafine fibers. Being a voltage free technique it can overcome the limitations of electrospinning. Owing to the unique characteristic features such as high surface area to volume ratio, porosity, mechanical strength and fiber alignment, centrifugal spun (C-spun) fibrous mat has a wide range of scope in various biomedical applications. Higher degree of fiber alignment can be effortlessly achieved by the C-Spin process. In order to prove the versatility of C-Spin system with respect to fiber alignment, Polycaprolactone (PCL) and gelatin were spun taking them as model polymers. The morphological analysis revealed that highly aligned ultrafine fibers with smooth surface are achieved by C-Spinning. Hydrophilicity, porosity and mechanical property results confirm that the C-spun mat is more suitable for tissue engineering applications. In vitro and in vivo experiments proved that the scaffolds are biocompatible and can be efficiently used as a wound dressing material.

Altered angiogenic balance in keloids: a key to therapeutic intervention.

Keloids are manifestations of abnormal wound repair with unresolved clinical complications. An effective therapeutic regimen has not been established for keloids, and current strategies are plagued by problems such as recurrence and side effects. Keloids, being a human-specific dermal fibroproliferative disorder are characterized by an excessive accumulation of extracellular matrix (ECM), thickened basement membrane, unregulated expression of matrix metalloproteases, growth factors, and cytokines. The internal milieu in a keloid bears a strong resemblance to a tumor with both exhibiting striking similarities with respect to tissue environment and unregulated vasculature. Abnormal angiogenesis manifested by an imbalance between proangiogenic and antiangiogenic factors has been recognized as a "common denominator" underlying many pathological conditions. However, such an imbalance has not been investigated in keloids. In this study, the angiogenic imbalance in keloids was explored with reference to circulating and tissue level expression of vascular endothelial growth factor (VEGF) and endostatin/collagen XVIII. It was observed that VEGF levels were upregulated and endostatin levels were downregulated in keloid patients in comparison to normal controls in both sera and tissue. Hence, antiangiogenic therapeutics based on endostatin in combination with current curative strategies as in tumors would present a scope for the effective management of keloids.