Claudia Chavez-Munoz

Profile of exosomes related proteins released by differentiated and undifferentiated human keratinocytes

Our group has previously demonstrated the capacity of human keratinocytes to release 14‐3‐3σ into conditioned medium through the mechanism of exosome externalization. In this study the release of other proteins through the same mechanism and the differences in the profiles of 14‐3‐3 proteins between differentiated (diff‐K) and undifferentiated keratinocytes (undiff‐K) were investigated. The stimulatory effect of other 14‐3‐3 isoforms on the expression of MMP‐1 in dermal fibroblasts was also evaluated. Exosomes isolated from undiff‐K (low Ca2+) and diff‐K (high Ca2+) were subjected to proteomic and Western blot analysis. The results showed that more than 50 different cytoplasmic proteins including all seven 14‐3‐3 protein isoforms (β, σ, η, ε, τ, ζ, and γ) were released from diff‐K through the mechanism of exosome externalization. However, in exosomes of undiff‐K only four of the 14‐3‐3 protein isoforms (β, η, ζ, and γ) were detected. Ca2+ treatment increased the release of exosomes from undiff‐K by at least two times relative to the control. Consistent with this finding, the stimulatory effect of exosomes containing 14‐3‐3σ from diff‐K had higher MMP‐1 stimulatory effect in fibroblasts relative to those exosomes isolated from undiff‐K. MMP‐1 stimulatory effect of recombinant 14‐3‐3β and η, tested in this study, in dermal fibroblasts, suggests additional anti‐fibrogenic factors other than 14‐3‐3σ. In conclusion, keratinocytes release many proteins through the mechanism of exosome externalization from which some such as 14‐3‐3 isoforms may function as extracellular matrix (ECM) modulating factors for dermal fibroblasts. These findings revealed the presence of a novel mechanism by which keratinocytes can potentially interact with fibroblasts. J. Cell. Physiol. 221: 221–231, 2009.

A novel hydrogel-collagen composite improves functionality of an injectable extracellular matrix

Cellular transplantation is now closer to becoming a practical clinical strategy to repair, regenerate or restore the function of skin, muscle, nerves and pancreatic islets. In this study we sought to develop a simple injectable collagen matrix that would preserve the normal cellular organization of skin cells. Three different scaffolds were created and compared: collagen-glycosaminoglycan (GAG) scaffolds, crosslinked collagen-GAG scaffolds without polyvinyl alcohol (PVA) and crosslinked collagen-GAG scaffolds containing PVA hydrogel. Importantly, all scaffolds were found to be non-cytotoxic. PVA-containing gels exhibited a higher tensile strength (P<0.05), faster fibril formation (P<0.001) and reduced collagenase digestion (P<0.01) compared with other gels. Free floating fibroblast-populated, PVA-borate scaffolds resisted contraction over a 10 day period (P<0.001). The fibroblast-populated scaffolds containing PVA demonstrated a 3-fold reduction in cellularity over 10 days compared with the control gels (P<0.001). Multicellular skin substitutes containing PVA-borate networks display a linear cellular organization, reduced cellularity and the formation of a keratinized epidermis that resembles normal skin. In conclusion, these data underscore the multifunctionality of a simple PVA-borate-collagen matrix as an injectable composite for tissue engineering or cell transplantation.

Primary human keratinocytes externalize stratifin protein via exosomes

Although, stratifin (SFN) is externalized by keratinocytes and stimulates the expression of matrix metalloproteinase‐1 (MMP‐1) in fibroblasts, its mechanism of externalization is not known. Here, we hypothesize that keratinocytes have a capacity to release stratifin through externalization of exosomes. To test this hypothesis, exosomes were purified from human keratinocyte conditioned medium (KCM) and analyzed for the presence of SFN by Western blot analysis using lysosomal‐associated membrane protein 2 (LAMP‐2) and heat shock cognate 70 (hsc70) as exosomal markers. The results showed the presence of SFN in keratinocyte lysate, concentrated KCM and exosomes, but not in concentrated unconditioned medium. Transmission electron microscopic examination revealed the presence of unique “saucer‐like” structures characteristic of exosomes whose diameters were <100 nm. Similar to the recombinant SFN, the exosomes associated proteins stimulated MMP‐1 expression in fibroblasts. Depletion of the exosomes markedly reduced this MMP‐1 stimulatory effect. To further statistically confirm these findings, fibroblasts were treated with three different exosome preparations and the finding showed more than 7.4‐fold increase in the level of MMP‐1 in the treated cells. Furthermore, we found that approximately 1% of the total proteins contained in exosomes correspond to SFN. In conclusion, this study is the first report showing that keratinocytes have the capacity to produce exosomes through which some intracellular proteins such as SFN, with MMP‐1 stimulating activity for fibroblasts, is externalized into keratinocyte microenvironment. J. Cell. Biochem. 104: 2165–2173, 2008.

Keratinocyte-releasable factors increased the expression of MMP1 and MMP3 in co-cultured fibroblasts under both 2D and 3D culture conditions

Matrix metalloproteinases (MMPs) are key elements in extracellular matrix (ECM) degradation and scar remodeling during the wound-healing process. Our previous data revealed that keratinocyte-releasable factors significantly increased the expression of fibroblast MMPs in monolayer-cultured fibroblasts. In this study, we analyzed the differences in the MMP expressions of fibroblasts in a three-dimensional fibroblast-populated collagen gel (3D FPCG) from that in a two-dimensional monolayer-cultured fibroblasts when both co-cultured with keratinocytes. Differential mRNA and protein expression of fibroblasts were examined by microarray, RT-PCR, and western blot. Our results showed that fibroblasts co-cultured with keratinocytes in a 3D FPCG expressed significantly higher MMP1 and MMP3 at the gene and protein levels. Due to the physiological advantages of a 3D FPCG model to a 2D system, we concluded that the 3D FPCG model may provide a better means of understanding the fibroblast–keratinocyte cross-talk during the wound-healing process.

Application of an Indoleamine 2,3-Dioxygenase–Expressing Skin Substitute Improves Scar Formation in a Fibrotic Animal Model

according to Prinsen et al., the cutoff scores for mildly, moderately, and severely impaired HRQoL on the emotions domain were X24, X35, and X39, respectively, meaning that a patient with a score X24 can be categorized as having a mildly impaired HRQoL on this domain, a score X35 as ‘‘moderate’’, etc. However, Samponga and Abeni categorized ‘‘mild’’ as having a score between 0 and 23.9 and, as a consequence, misclassified all cutoff scores. Therefore, we would like to provide a correct overview of the categorization of Skindex-29 scores (Table 1). Having said this, we fully agree with Sampogna and Abeni on the limitations of both methods, such as dependence on the distribution of HRQoL scores in estimation samples and biases when using prospective anchors. Nevertheless, we believe that, under the condition that the same scale or anchor question is being used, anchor-based methods may lead to less variant estimates of cutoff scores than distribution-based methods. In addition, anchor-based methods are less dependent on the sociocultural and clinical characteristics of the estimation sample. For example, patients in one sample, scoring themselves as having a severely impaired HRQoL on a global rating scale or anchor question (for instance, an anchor question such as ‘‘In your opinion, how severe is your skin condition?’’), are likely to have Skindex-29 scores in the same range of scores as patients of another sample who also score themselves as having a severely impaired HRQoL. Nevertheless, the phrasing of an anchor question is a great source of variation in the comparison of different cutoff scores. We therefore advocate the use of standardized anchors. A clinically meaningful interpretation of Skindex-29 scores is of great value. At present, two studies on this intriguing subject are available. As already expressed by Sampogna and Abeni, the combination of an anchorbased and a distribution-based method in a subsequent study would allow an objective comparison of the results within one study population. In addition to this, we recommend including standardized anchors, and to conduct such a study on an international level. Eventually, such efforts will contribute to reaching consensus on the categorization of scores so that they can be applied in clinical practice.

Dermal fibroblasts influence the expression profile of 14-3-3 proteins in human keratinocytes

We have previously demonstrated that the release of some of the 14-3-3 isoforms from keratinocytes is able to influence the expression of key matrix metalloproteinases (MMPs) in dermal fibroblasts. Conversely, in this study we aimed to investigate whether dermal fibroblasts possess the ability to modulate the expression of 14-3-3 proteins in keratinocytes. In order to address this question, human keratinocytes and dermal fibroblasts were harvested and co-cultured. Intra- and extracellular levels of 14-3-3 proteins (β, η, γ, and σ) were analyzed using western blot analysis, and the gene expression was further assessed by quantitative real-time polymerase chain reaction. Gene analysis revealed an up-regulation of all four 14-3-3 isoforms of interest. In addition, the findings of this study reveal a significant increase in the intracellular levels of 14-3-3 γ and σ in keratinocytes co-cultured with fibroblasts compared to those of the mono-cultured control keratinocytes. Mechanistic investigations also demonstrated the capacity of several mitogen-activated protein kinase-specific inhibitors to markedly reduce induction of 14-3-3 σ in keratinocytes stimulated with fibroblast-conditioned medium. The study concluded that dermal fibroblasts possess the ability to influence the expression of several 14-3-3 isoforms (notably γ and σ) in keratinocytes, suggesting that the two cell types might be capable of bi-directionally influencing the protein expression of one another in vivo.

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase‐1) and type I collagen in fibroblasts. The first one, we called it keratinocyte‐derived anti‐fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14‐3‐3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte‐derived collagen‐inhibiting factor(s) (KD‐CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte‐conditioned media. Using co‐immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid. J. Cell. Biochem. 113: 2622–2632, 2012.

Calcium-sensing receptor (CaSR) promotes development of bone metastasis in renal cell carcinoma

Bone metastasis is an important prognostic factor in renal cell carcinoma (RCC). The calcium-sensing receptor (CaSR) has been associated with bone metastasis in several different malignancies. We analyzed the impact of CaSR in bone metastasis in RCC in vitro and in vivo. The RCC cell line 786-O was stably transfected with the CaSR gene and treated with calcium alone or in combination with the CaSR antagonist NPS2143. Afterwards migration, adhesion, proliferation and prominent signaling molecules were analyzed. Calcium treated CaSR-transfected 768-O cells showed an increased adhesion to endothelial cells and the extracellular matrix components fibronectin and collagen I, but not to collagen IV. The chemotactic cell migration and proliferation was also induced by calcium. The activity of SHC, AKT, ERK, P90RSK and JNK were enhanced after calcium treatment of CaSR-transfected cells. These effects were abolished by NPS2143. Development of bone metastasis was evaluated in vivo in a mouse model. Intracardiac injection of CaSR-transfected 768-O cells showed an increased rate of bone metastasis. The results indicate CaSR as an important component in the mechanism of bone metastasis in RCC. Therefore, targeting CaSR might be beneficial in patients with bone metastatic RCC with a high CaSR expression.

Magnetically-actuated drug delivery device (MADDD) for minimally invasive treatment of prostate cancer: An in vivo animal pilot study

The vast majority of prostate cancer presents clinically localized to the prostate without evidence of metastasis. Currently, there are several modalities available to treat this particular disease. Despite radical prostatectomy demonstrating a modest prostate cancer specific mortality benefit in the PIVOT trial, several novel modalities have emerged to treat localized prostate cancer in patients that are either not eligible for surgery or that prefer an alternative approach.

Identification of hypoxic gene-signature as a prognostic and predictive biomarker to determine effective therapy in high risk bladder cancer patients

Bladder cancer is the ninth most commonly diagnosed cancer in the world and fth in the USA and Canada (1,2). Patients presenting with bladder cancer are initially treated with transurethral resection and specimens are graded and staged.