Clara Chamorro - Academia.edu (original) (raw)

Papers by Clara Chamorro

The biology of epithelial immunity as well as of tissue repair is complex and highly regulated. A... more The biology of epithelial immunity as well as of tissue repair is complex and highly regulated. A variety of molecules, cell types, and biological processes such as, cell differentiation, proliferation and migration, programmed cell death and antimicrobial mechanisms contribute to maintaining a balance between tissue damage and tissue repair. Different sources of evidence indicated that the cathelicidin hCAP18/LL-37 might, not only act as an antibacterial molecule, but as a key regulator of the above mentioned processes. We therefore tested this concept through in vivo and in vitro studies aimed at determining whether and how hCAP18/LL-37 could be such a multifunctional molecule. The goal of this thesis was to investigate the role of LL-37 in epithelial cell biology by addressing the following questions: How is the expression of the human cathelicidin gene (CAMP) controlled in primary keratinocytes? Does LL-37 contribute to tissue repair through either, regulation of cell proliferation, differentiation and/or programmed cell death? Which genes become expressed in the presence of LL-37 and what potential biological processes do they control? And does LL-37 contribute to defects in cell proliferation, differentiation and/or migration during pathological states such as cancer? We found that the active form of Vitamin D (VD) and its metabolites induced the expression of CAMP. As the biological effects of VD are mediated by the vitamin D receptor (VDR), which, once activated binds to response elements in the promoter region of target genes (VDRE), we tested the CAMP promoter for the presence of VDR binding sites. We identified one active VDRE binding site at about ~500 bp from the transcription start site. In vivo stimulation of human skin with the vitamin D analog calcipotriol resulted in high expression of hCAP18 at the mRNA and protein levels, compared to control skin samples from the same individuals (Paper I). Tissue homeostasis is maintained by, among others, the selective removal of cells through mechanisms such as apoptosis or programmed cell death. Because cell proliferation and differentiation are central to skin biology, we tested the role of LL-37 in the apoptosis of human keratinocytes. We found that LL-37 prevented camptothecin-induced apoptosis, which was associated with decreased caspase-3 activity and increased expression of PGE2 and IAP2, through a mechanism dependent on COX2 activity (Paper II). By using a microarray approach we found that LL-37 affected the gene expression profile of human keratinocytes with a signifficant effect on STC2. We investigated the mechanism of LL-37-induced STC2 upregulation and found evidence (paper III, manuscript) suggesting that unfolded protein response in keratinocytes might be triggered by expossure to LL-37. We measured the expression of the hCAP18/LL-37 in a panel of 104 breast cancer tumors and compared it to the levels found in control samples, as well as analyzed its relationship to clinical data. We also studied, in vitro, the effect of LL-37 on breast cancer cell migration and in anchorage independent colony formation. In addition, we explored the molecular mechanisms responsible for LL-37 effects on breast cancer cells. To evaluate the relevance of our findings in vivo, a xenograft model using severely compromised immunodeficient (SCID) mice was designed to test the effect of the transgenic expression of the hCAP18/LL-37 in tumor formation. The results from these experiments indicate that 1) hCAP18/LL-37 is functionally connected with ErbB2; 2) LL-37 alters breast cancer cell phenotype in vitro 3) LL-37 stimulates the migration of breast cancer cells and 4) LL-37 stimulates metastasis formation in SCDI mice in vivo (paper IV). Increasing evidence shows that besides its immune function, LL-37 has tissue-repair-like effects, promotes cell proliferation, migration and angiogenesis. Furthermore LL-37 has been implicated in the pathogenesis of inflammatory skin disease such as psoriasis. Based on our findings, we propose that LL-37 is a key regulator of epithelial homeostasis by influencing, tissue defense, tissue repair and maintenance through control of programmed cell death, in association with vitamin D and likely acting as an alarmin by activating processes such as the unfolded protein response. Thus, these effects become relevant for the study of pathological states such as chronic inflammation and cancer.

Journal of Tissue Science and Engineering, 2016

Congenital defects of the urinary bladder that requires surgical intervention with mechanical wou... more Congenital defects of the urinary bladder that requires surgical intervention with mechanical wounding are common situations among pediatric urology patients. In conditions with severe lack of tissue, regenerative medicine with autologous cells has become a field of interest for future cure. In both situations, normal bladder wound healing is of major importance for an uneventful healing process and for the final results. Much effort has been put into increasing our understanding in the area of urinary bladder wound healing. Several methods have been used in different studies, all representing different clinical settings to address the issue of normal healing. However, little is known about the differences between these different wound-healing models. In this review, we aimed at summarizing what is known about the process of bladder wound healing after mechanical injury. We present the most commonly used methods in this area; describe the process of healing and the current knowledge on involved signaling transduction factors.

Frontiers in Pediatrics, Jun 22, 2021

Conclusions: The importance of this study is that we used a chronic large urethral defect animal ... more Conclusions: The importance of this study is that we used a chronic large urethral defect animal model and clearly found that cell-seeded transplants were superior to nonseeded. In addition, bladder washing was a feasible method for harvesting viable autologous cells in a noninvasive way. There is a place for considering tissue-engineered transplants in the surgical armamentarium for treating complex urethral defects and hypospadias cases.

Journal of Pediatric Urology, Apr 1, 2023

 Users may download and print one copy of any publication from the public portal for the purpose... more  Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Anais Brasileiros De Dermatologia, Dec 1, 2009

Journal of Tissue Engineering and Regenerative Medicine, Jul 22, 2019

The success of regenerative medicine relies in part on the quality of the cells implanted. Cell c... more The success of regenerative medicine relies in part on the quality of the cells implanted. Cell cultures from cells isolated from bladder washes have been successfully established but molecular changes and cell characteristics have not been explored in detail. In this work, we analysed the role of telomere shortening in relation to the regenerative potential and senescence of cells isolated form bladder washes and expanded in culture. We also analysed, whether bladder washes would be a potential source for attaining stem cells or promoting stem cell proliferation by using two different substrates to support their growth: a feeder layer of growth arrested murine fibroblasts J2 3T3 cells and a xeno-free human recombinant laminin coated surface. We found no association between telomere shortening and senescence in urothelial cells in vitro. Urothelial cells had a stable telomere length and expressed mesenchymal stem cells markers but failed to differentiate into bone or adipocytes. Feeder layer showed an advantage to laminin-coated surfaces in respect to proliferative capacity with the expense of risking that feeder layer cells could persist in later passages. This emphasizes the importance of using carefully controlled culture conditions and molecular quality controls before autotransplantation in future clinical settings. In conclusion, urothelial cells isolated by bladder washes show regenerative potential that need further understanding. Senescence in vitro might be due to cellular stress and, if so,

DOAJ (DOAJ: Directory of Open Access Journals), Dec 1, 2009

En las últimas tres décadas el cultivo y proliferación de queratinocitos humanos ha emergido como... more En las últimas tres décadas el cultivo y proliferación de queratinocitos humanos ha emergido como una herramienta poderosa,

Scientific Reports, Nov 8, 2021

Urinary bladder wound healing relies on multiple biological events that are finely tuned in a spa... more Urinary bladder wound healing relies on multiple biological events that are finely tuned in a spatialtemporal manner. MicroRNAs are small non-coding RNA molecules with regulatory functions. We hypothesized that microRNAs are important molecules in the coordination of normal urinary bladder wound healing. We aimed at identifying microRNAs expressed during bladder wound healing using Affymetrix global array for microRNA profiling of the rodent urinary bladder during healing of a surgically created wound. Results were validated in the rat bladders by real-time PCR (RT-PCR) using three of the differentially expressed (DE) microRNAs. The model was thereafter validated in human cells, by measuring the expression of eight of the DE microRNAs upon in vitro wound-healing assays in primary urothelial cells. Our results indicated that 508 (40%) of all rodent microRNAs were expressed in the urinary bladder during wound healing. Thirteen of these microRNAs (1%) were DE (false discovery rate (FDR) < 0.05, P < 0.05, |logfold|> 0.25) in wounded compared to non-wounded bladders. Bioinformatic analyses helped us to identify target molecules for the DE microRNAs, and biological pathways involved in tissue repair. All data are made available in an open-access database for other researchers to explore. Engineered cell-seeded grafts have been introduced to treat several urogenital disorders 1,2. The final outcome of any transplanted tissue or engineered graft greatly depends on the host wound healing response. Wound healing in adult tissue is a multistep process comprising overlapping cellular responses including hemostasis, inflammation, cellular proliferation, extracellular matrix synthesis and remodeling. In a normal wound, once the hemostasis and inflammation steps have ensued, proliferation and cellular migration take over and replace the damaged tissue. Eventually, the proliferation ceases and the tissue matures with scar formation. In the rodent urinary bladder, the healing process is highly similar to what has been observed in the skin, at both a morphological and a molecular level 3,4. Due to their reliability and reproducibility, rodent models are frequently used in biomedical research 5. In comparison to the human genome, the rat genome encodes a similar number of protein-coding genes (22,000 for rats, 20,000 for humans) 5,6. Molecular and developmental processes in rodents are well characterized and share great similarities with humans 7. Similarly, healing and trauma models in rats have proven translationally valuable for human wound healing and trauma research 8-11. The knowledge about the molecular mechanisms that coordinate bladder healing may be highly relevant to the clinical practice. It may help us understand conditions, such as chronic cystitis and post-irradiation cystitis, and it may improve the surgical treatments of congenital conditions, such as bladder exstrophy. Further understanding of micro-molecular events may unlock the development of pharmaceuticals for treating the diseased urinary bladder. The non-coding small RNA molecules (19-23 nucleotides long), known as microRNAs, are important players during tissue homeostasis. These molecules act as epigenetic regulators of gene expression at the post-transcriptional level 12. Mature microRNA molecules can target the degradation of complementary messenger RNAs (mRNA) or by inhibiting the interaction between mRNA and the translational machinery (down-regulating control) 13. MicroRNAs interact mainly with the 3'untranslated region of target mRNAs, but the molecules can

International Journal of Biological Macromolecules, Aug 1, 2022

Autologous micrografting is a technique currently applied within skin wound healing, however, the... more Autologous micrografting is a technique currently applied within skin wound healing, however, the potential use for surgical correction of other organs with epithelial lining, including the urinary bladder, remains largely unexplored. Currently, little is known about the micrograft expansion potential and the micromolecular events that occur in micrografted urothelial cells. In this study, we aimed to evaluate the regenerative potential of different porcine urothelial micrograft sizes in vitro, and, furthermore, to explore how urothelial micrografts communicate and which microcellular events are triggered. We demonstrated that increased tissue fragmentation subsequently potentiated the yield of proliferative cells and the cellular expansion potential, which confirms, that the micrografting principles of skin epithelium also apply to uroepithelium. Furthermore, we targeted the expression of the extracellular signal-regulated kinase (ERK) pathway and demonstrated that ERK activation o...

Frontiers in Pediatrics

Introduction: Tissue engineering is a potential source of urethral substitutes to treat severe ur... more Introduction: Tissue engineering is a potential source of urethral substitutes to treat severe urethral defects. Our aim was to create tissue-engineered urethras by harvesting autologous cells obtained by bladder washes and then using these cells to create a neourethra in a chronic large urethral defect in a rabbit model.Methods: A large urethral defect was first created in male New Zealand rabbits by resecting an elliptic defect (70 mm2) in the ventral penile urethra and then letting it settle down as a chronic defect for 5–6 weeks. Urothelial cells were harvested noninvasively by washing the bladder with saline and isolating urothelial cells. Neourethras were created by seeding urothelial cells on a commercially available decellularized intestinal submucosa matrix (Biodesign® Cook-Biotech®). Twenty-two rabbits were divided into three groups. Group-A (n = 2) is a control group (urethral defect unrepaired). Group-B (n = 10) and group-C (n = 10) underwent on-lay urethroplasty, with u...

Revista argentina de …, 2009

International Journal of Molecular Sciences, Oct 21, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Scientific Reports, 2021

International Journal of Biological Macromolecules

Tissue Engineering Part A, 2015

Cultured human urothelial cells can be used in tissue engineering for reconstruction of urothelia... more Cultured human urothelial cells can be used in tissue engineering for reconstruction of urothelial defects. For safety reasons, a fine characterization of the cells is required before its use in reconstructive surgery. For these reasons we aimed to characterize the effect of in vitro propagation of urothelial cells on gene expression and proliferative capacity. Gene expression of urothelial cells in passage two and eight were captured by using a microarray chip covering the whole human genome. In order to find relationships in biological functions and pathways differentially regulated genes were subjected to pathway analysis using the WEB-based Gene Set Analysis Toolkit (WebGestalt). Proliferative capacity was tested with population doubling time, efficiency in colony formation assays and immunocytochemistry. In addition senescence markers were evaluated. Bioinformatics' analysis reveled gene expression profile differences. Down-regulated genes at passage eight clustered in biological pathways of cell cycle and DNA repair processes; up regulated genes had no obvious association to any specific biological function or pathway according to WebGestalt analysis, but individual genes with extracellular matrix, apoptosis and cell morphology. Data was supported by RT-PCR and in vitro growth experiments. Passage two urothelial cells had higher efficiency in colony formation and lower population doubling time. An increase in senescence markers was detected at passage eight. We conclude that pre-transplantation quality controls are important and, for reconstructive purposes, cells should be transplanted back to the patient as soon as possible to procure good proliferative capacity also after transplantation.

International Journal of Cancer, 2004

Human cathelicidin antimicrobial protein hCAP18/LL-37 is an effector molecule of the nonspecific ... more Human cathelicidin antimicrobial protein hCAP18/LL-37 is an effector molecule of the nonspecific innate immune system. hCAP18/LL-37 is present in leukocytes and is expressed in skin and other epithelia, where it is upregulated in association with inflammation and injury. In addition, antimicrobial proteins including cathelicidins have been proposed to play a role in the nonspecific defense against tumors. To assess its potential role in tumor host defense, we investigated the expression of hCAP18/LL-37 in a series of breast carcinomas. Unexpectedly, we found that hCAP18/LL-37 was strongly expressed in the tumor cells and not in the adjacent stroma. To test the hypothesis that hCAP18/LL-37 may provide a growth advantage for the tumor cells, we treated human epithelial cell lines with synthetic biologically active LL-37 peptide and found a significant increase in cell proliferation. In addition, transgenic expression of hCAP18 in 2 different human epithelial cell lines resulted in increased proliferation of both cell types. These findings do not support the hypothesis that LL-37 has an antitumor effect, but rather suggest that hCAP18/LL-37 may promote tumor cell growth in breast cancer.

Breast Cancer Research, 2009

Introduction Human cathelicidin antimicrobial protein, hCAP18, and its C-terminal peptide LL-37 i... more Introduction Human cathelicidin antimicrobial protein, hCAP18, and its C-terminal peptide LL-37 is a multifunctional protein. In addition to being important in antimicrobial defense, it induces chemotaxis, stimulates angiogenesis and promotes tissue repair. We previously showed that human breast cancer cells express high amounts of hCAP18, and hypothesised that hCAP18/LL-37 may be involved in tumour progression. Methods hCAP18 mRNA was quantified in 109 primary breast cancers and compared with clinical findings and ERBB2 mRNA expression. Effects of exogenous LL-37 and transgenic overexpression of hCAP18 on ErbB2 signalling were investigated by immunoblotting using extracts from breast cancer cell lines ZR75-1 and derivatives of MCF7. We further analysed the impact of hCAP18/LL-37 on the morphology of breast cancer cells grown in soft agar, on cell migration and on tumour development in severe combined immunodeficiency (SCID) mice. Results The expression of hCAP18 correlated closely with that of ERBB2 and with the presence of lymph node metastases in oestrogen receptor-positive tumours. hCAP18/LL-37 amplified Heregulin-induced mitogen-activated protein kinase (MAPK) signalling through ErbB2, identifying a functional association between hCAP18/LL-37 and ErbB2 in breast cancer. Treatment with LL-37 peptide significantly stimulated the migration of breast cancer cells and their colonies acquired a dispersed morphology indicative of increased metastatic potential. A truncated version of LL-37 competitively inhibited LL-37 induced MAPK phosphorylation and significantly reduced the number of altered cancer cell colonies induced by LL-37 as well as suppressed their migration. Transgenic overexpression of hCAP18 in a low malignant breast cancer cell line promoted the development of metastases in SCID mice, and analysis of hCAP18 transgenic tumours showed enhanced activation of MAPK signalling. Conclusions Our results provide evidence that hCAP18/LL-37 contributes to breast cancer metastasis.

Journal of Investigative Dermatology, 2005