Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization.

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

Local Probiotic Therapy with Lactobacillus plantarum Mitigates Scar Formation in Rabbits after Burn Injury and Infection.

BACKGROUND: Infection is the most common complication in burn-injured patients and is believed to contribute to the hypertrophic scarring frequently observed in such injury. Pseudomonas aeruginosa is a common pathogen in burn wound infection. We examined the effect of local probiotic therapy with Lactobacillus plantarum on the severity of the scarring following burn wounding and infection with P. aeruginosa in a rabbit model. METHODS: Full-thickness burn wounds were inoculated with control vehicle or L. plantarum; wounds were then challenged with bioluminescent P. aeruginosa. The time course of the ensuing infection was monitored by quantification of the emitted light. After allowing wounds to contract to near completion, they were harvested and analyzed for markers of scar formation. RESULTS: Application of L. plantarum curtailed both the severity and the length of the pseudomonal infection. Probiotic therapy significantly reduced both Type I collagen mRNA concentrations and total collagen protein accumulation in infected wounds, consistent with reduced scarring. Surprisingly, the probiotic showed a nearly equivalent effect in uninfected wounds. Masson's trichrome staining confirmed these findings histologically. CONCLUSIONS: Lactobacillus plantarum shows exciting potential as a therapeutic agent to both counteract burn wound infection and to alleviate scarring even in the absence of infection.

Anti-fibrotic action of pirfenidone in Dupuytren's disease-derived fibroblasts.

BACKGROUND: Dupuytren's disease (DD) is a complex fibro-proliferative disorder of the hand that is often progressive and eventually can cause contractures of the affected fingers. Transforming growth factor beta (TGF-ß1) has been implicated as a key stimulator of myofibroblast activity and fascial contraction in DD. Pirfenidone (PFD) is an active small molecule shown to inhibit TGF-ß1-mediated action in other fibrotic disorders. This study investigates the efficacy of PFD in vitro in inhibiting TGF-ß1-mediated cellular functions leading to Dupuytren's fibrosis. METHODS: Fibroblasts harvested from (DD) and carpal tunnel (CT)- tissues were treated with or without TGF-ß1 and/or PFD and were subjected to cell migration, cell proliferation and cell contraction assays. ELISA; western blots and real time RT-PCR assays were performed to determine the levels of fibronectin; p-Smad2/Smad3; alpha-smooth muscle actin (α-SMA), α2 chain of type I collagen and α1 chain of type III collagen respectively. RESULTS: Our results show that PFD effectively inhibits TGF-ß1-induced cell migration, proliferation and cell contractile properties of both CT- and DD-derived fibroblasts. TGF-ß1-induced α-SMA mRNA and protein levels were inhibited at the higher concentration of PFD (800 µg/ml). Interestingly, TGF-ß1 induction of type I and type III collagens and fibronectin was inhibited by PFD in both CT- and DD- derived fibroblasts, but the effect was more prominent in DD cells. PFD down-regulated TGF-ß1-induced phosphorylation of Smad2/Smad3, a key factor in the TGF-ß1 signaling pathway. CONCLUSION: Taken together these results suggest the PFD can potentially prevent TGF-ß1-induced fibroblast to myofibroblast transformation and inhibit ECM production mainly Type I- and Type III- collagen and fibronectin in DD-derived fibroblasts. Further in-vivo studies with PFD may lead to a novel therapeutic application in preventing the progression or recurrence of Dupuytren's disease.

Expression analysis of human adipose-derived stem cells during in vitro differentiation to an adipocyte lineage.

BACKGROUND: Adipose tissue-derived stromal stem cells (ASCs) represent a promising regenerative resource for soft tissue reconstruction. Although autologous grafting of whole fat has long been practiced, a major clinical limitation of this technique is inconsistent long-term graft retention. To understand the changes in cell function during the transition of ASCs into fully mature fat cells, we compared the transcriptome profiles of cultured undifferentiated human primary ASCs under conditions leading to acquisition of a mature adipocyte phenotype. METHODS: Microarray analysis was performed on total RNA extracted from separate ACS isolates of six human adult females before and after 7 days (7 days: early stage) and 21 days (21 days: late stage) of adipocyte differentiation in vitro. Differential gene expression profiles were determined using Partek Genomics Suite Version 6.4 for analysis of variance (ANOVA) based on time in culture. We also performed unsupervised hierarchical clustering to test for gene expression patterns among the three cell populations. Ingenuity Pathway Analysis was used to determine biologically significant networks and canonical pathways relevant to adipogenesis. RESULTS: Cells at each stage showed remarkable intra-group consistency of expression profiles while abundant differences were detected across stages and groups. More than 14,000 transcripts were significantly altered during differentiation while ~6000 transcripts were affected between 7 days and 21 days cultures. Setting a cutoff of +/-two-fold change, 1350 transcripts were elevated while 2929 genes were significantly decreased by 7 days. Comparison of early and late stage cultures revealed increased expression of 1107 transcripts while 606 genes showed significantly reduced expression. In addition to confirming differential expression of known markers of adipogenesis (e.g., FABP4, ADIPOQ, PLIN4), multiple genes and signaling pathways not previously known to be involved in regulating adipogenesis were identified (e.g. POSTN, PPP1R1A, FGF11) as potential novel mediators of adipogenesis. Quantitative RT-PCR validated the microarray results. CONCLUSIONS: ASC maturation into an adipocyte phenotype proceeds from a gene expression program that involves thousands of genes. This is the first study to compare mRNA expression profiles during early and late stage adipogenesis using cultured human primary ASCs from multiple patients.

Cloning of TgfßR1 and TgfßR2 and Likely Exclusion as Loci of Origin in a Rabbit Craniosynostotic Model.

OBJECTIVE: To determine whether TgfßR1 or TgfßR2 cause the craniosynostotic phenotype in a rabbit model of nonsyndromic craniosynostosis. DESIGN: Full-length TgfßR1 and TgfßR2 cDNAs were sequenced and real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed to measure TgfßR1 and TgfßR2 transcripts in sutural tissue from wild type (WT) and craniosynostotic (CS) rabbits. Single nucleotide polymorphisms (SNP) were identified within TgfßR1 and TgfßR2 and were assayed for segregation with disease phenotype in 22 craniosynostotic animals. RESULTS: No structural mutations in TgfßR1 and TgfßR2 were identified in the craniosynostotic rabbits. Real-time RT-PCR quantification of TgfßR1 and TgfßR2 mRNA showed no significant difference in TgfßR1 expression between CS and WT animals, while TgfßR2 showed 50% elevation in the CS animals compared to WT (P < .05). SNP analysis within the TgfßR1 and TgfßR2 genes suggested that neither locus is linked to the craniosynostotic phenotype because no allelic combination showed any specific correlation with disease phenotype for either TgfßR1 or TgfßR2. CONCLUSIONS: Our data indicate that the craniosynostotic phenotype in this rabbit model does not arise from any structural mutation in TgfßR1 or TgfßR2, and SNP analysis also likely excludes these genes more broadly as the site of causative mutation.

Molecular Analysis of Twist1 and FGF Receptors in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Craniosynostosis is the premature fusion of the cranial vault sutures. We have previously described a colony of rabbits with a heritable pattern of nonsyndromic, coronal suture synostosis; however, the underlying genetic defect remains unknown. We now report a molecular analysis to determine if four genes implicated in human craniosynostosis, TWIST1 and fibroblast growth factor receptors 1-3 (FGFR1-3), could be the loci of the causative mutation in this unique rabbit model. Single nucleotide polymorphisms (SNPs) were identified within the Twist1, FGFR1, and FGFR2 genes, and the allelic patterns of these silent mutations were examined in 22 craniosynostotic rabbits. SNP analysis of the Twist1, FGFR1, and FGFR2 genes indicated that none were the locus of origin of the craniosynostotic phenotype. In addition, no structural mutations were identified by direct sequence analysis of Twist1 and FGFR3 cDNAs. These data indicate that the causative locus for heritable craniosynostosis in this rabbit model is not within the Twist1, FGFR1, and FGFR2 genes. Although a locus in intronic or flanking sequences of FGFR3 remains possible, no direct structural mutation was identified for FGFR3.

Scarless integumentary wound healing in the mammalian fetus: molecular basis and therapeutic implications.

Adult mammals respond to injury of their skin/integument by forming scar tissue. Scar is useful in rapidly sealing an injured area, but can also lead to significant morbidity. Mammals in fetal life retain the ability to heal integumentary wounds regeneratively, without scar. The critical molecular mechanisms governing this remarkable phenomenon have been a subject of great interest, in the hopes that these could be dissected and recapitulated in the healing adult wound, with the goal of inducing scarless healing in injured patients. Multiple lines of investigation spanning decades have implicated a number of factors in distinguishing scarless from fibrotic wound healing, including most prominently transforming growth factor-ß and interleukin-10, among others. Therapeutic interventions to try to mitigate scarring in adult wounds have been developed out of these studies, and have reached the level of clinical trials in humans, although as yet no FDA-approved treatment exists. More recent expressomic studies have revealed many more genes that are differentially expressed in scarlessly healing fetal wounds compared with adult, and microRNAs have also been identified as participating in the fetal wound healing response. These represent an even greater range of potential therapeutics (or targets for therapy) to translate the promise of scarless fetal wound healing to the injured adult patient.

Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture.

BACKGROUND: Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. METHODS: To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram. RESULTS: We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells. In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder. CONCLUSIONS: These data show that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected palmar fascia in DC patients are highly similar, and differ significantly from the transcriptomic profiles of fibroblasts from the palmar fascia of patients undergoing carpal tunnel release.

Molecular analysis of coronal perisutural tissues in a craniosynostotic rabbit model using polymerase chain reaction suppression subtractive hybridization.

BACKGROUND: In the United States, the incidence of craniosynostosis (premature fusion of the sutures of the cranial vault) is one in 2000 to 3000 live births. The condition can cause increased intracranial pressure, severely altered head shape, and mental retardation. The authors have previously described a colony of rabbits with heritable coronal suture synostosis. This model has been instrumental in describing the postsurgical craniofacial growth associated with craniosynostosis. The molecular analysis of this model has been limited by the lack of molecular tools for use in rabbits. To understand the pathogenesis of craniosynostosis, the authors compared gene expression in perisutural tissues between wild-type and craniosynostotic rabbits using polymerase chain reaction suppression subtractive hybridization. METHODS: Suppression subtractive hybridization polymerase chain reaction was performed on RNA derived from pooled samples of calvariae from 10-day-old wild-type (n = 3) and craniosynostotic (n = 3) rabbits to obtain cDNA clones enriched in either wild-type tissues (underexpressed in craniosynostotic tissue) or craniosynostotic tissues (overexpressed in craniosynostotic compared with wild-type). RESULTS: Differential expression was identified for approximately 140 recovered cDNA clones up-regulated in craniosynostotic tissues and 130 recovered clones for wild-type tissues. Of these, four genes were confirmed by quantitative reverse-transcriptase polymerase chain reaction as being overexpressed in craniosynostotic sutural tissue: ß-globin (HBB), osteopontin (SPP1), osteonectin (SPARC), and cathepsin K (CTSK). Two genes were confirmed to be underexpressed in the craniosynostotic samples: collagen 3, alpha 1 (COL3A1) and ring finger protein 12 (RNF12). CONCLUSION: The differential expression of these gene products in our naturally occurring craniosynostotic model appears to be the result of differences in the normal bone formation/resorption pathway.

Increased expression of Ero1L-alpha in healing fetal wounds.

BACKGROUND: Adult mammalian tissues heal injury to the skin with formation of scar; this process quickly seals an injured area, however, excessive scar formation can become a source of persistent pathology, interfering with multiple vital functions. In contrast, mammalian fetal tissue can heal without scar formation. We previously sought to model scarless healing in a rabbit fetal skin wound and identified gene products differentially expressed during fetal wound healing through PCR suppression subtractive hybridization (PCR SSH). One of these transcripts, previously identified simply as clone 11, showed putative increased expression in wounded fetal skin. This study establishes its identity as Ero1L-alpha and confirms its elevated expression in healing fetal wounds. FINDINGS: After obtaining further sequence by 5' rapid amplification of cloned ends (RACE) we find that clone 11 is Ero1L-alpha. We determined that clone 11, a differentially expressed transcript in fetal wound healing, comprises the 3' untranslated region (UTR) of an approximately 4 kb transcript in rabbit tissues that corresponds to Ero1L-alpha. We showed that Ero1L-alpha is expressed predominantly as two transcripts in rabbit skin, namely a 1.6 kb transcript and the 4.0 kb transcript recovered in our PCR SSH screen via its 3' UTR sequence. However, a third transcript of 2.9 kb was also detected in Northern blots and was subsequently cloned and confirmed by 3' RACE. Knockdown of the clone 11 sequence in rabbit adult fibroblasts via siRNA resulted in significantly decreased Ero1L-alpha message expression. Increased expression of clone 11 (Ero1L-alpha) in a variety of cell types during the wound healing response was also confirmed by in situ hybridization. CONCLUSIONS: Ero1L-alpha is one of the previously unknown clones identified in a PCR SSH screen for genes differentially expressed in fetal wounded tissue. In situ hybridization confirms that Ero1L-alpha shows increased expression in multiple cell types after wounding of the fetal integument.

Chaperonin containing T-complex polypeptide (CCT) subunit expression in oral mucosal wounds and fibroblasts.

Mucosal wound healing in adults has been reported to feature diminished scar formation compared to healing skin wounds. We sought to determine if the expression pattern of chaperonin containing T-complex polypeptide (CCT) subunits in mucosal wounds and fibroblasts is different from that observed in skin wounds and fibroblasts. We found that CCT-beta is the only subunit message to be reduced in wounded mucosa versus unwounded control, and this reduction was confirmed at the protein level. In contrast, mRNA levels of CCT-zeta, -delta, -eta, and -epsilon were significantly increased in mucosal wounds. The increase in CCT-eta was also confirmed at the protein level. Expression levels of CCT-alpha, -beta, -delta; -epsilon, and -theta mRNAs were significantly increased in adult mucosal fibroblasts in culture compared to skin-derived fibroblasts. Western blot analyses confirmed a modest increase in CCT-beta in adult mucosal fibroblasts relative to skin fibroblasts, but CCT-eta protein was unaffected. These differences may contribute to the reported difference in healing outcomes between these two tissue types.

Reversal of TGF-ß1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's-derived fibroblasts.

BACKGROUND: Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha- smooth muscle actin (α-SMA) and increased production of extracellular matrix (ECM) components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic AMP (cAMP)/protein kinase A signaling pathway will inhibit transforming growth factor-beta1 (TGF-ß1)-induced ECM synthesis and myofibroblast formation and may provide a means to blunt fibrosis. METHODS: Fibroblasts derived from areas of Dupuytren's contracture cord (DC), from adjacent and phenotypically normal palmar fascia (PF), and from palmar fascia from patients undergoing carpal tunnel release (CTR; CT) were treated with TGF-ß1 (2 ng/ml) and/or forskolin (10 µM) (a known stimulator of cAMP). Total RNA and protein extracted was subjected to real time RT-PCR and Western blot analysis. RESULTS: The basal mRNA expression levels of fibronectin- extra domain A (FN1-EDA), type I (COL1A2) and type III collagen (COL3A1), and connective tissue growth factor (CTGF) were all significantly increased in DC- and in PF-derived cells compared to CT-derived fibroblasts. The TGF-ß1 stimulation of α-SMA, CTGF, COL1A2 and COL3A1 was greatly inhibited by concomitant treatment with forskolin, especially in DC-derived cells. In contrast, TGF-ß1 stimulation of FN1-EDA showed similar levels of reduction with the addition of forskolin in all three cell types. CONCLUSION: In sum, increasing cAMP levels show potential to inhibit the formation of myofibroblasts and accumulation of ECM components. Molecular agents that increase cAMP may therefore prove useful in mitigating DC progression or recurrence.

Generation of transgenic mice expressing Cre recombinase under the control of the Dll1 mesoderm enhancer element.

To study paraxial mesoderm formation in the mouse, transgenic lines that can be used to either selectively delete or express genes of interest in the paraxial mesoderm are required. We have generated a transgenic mouse line that expresses Cre recombinase in the paraxial mesoderm (PAM) beginning at e7.5. A lacZ Cre recombinase reporter line showed that in addition to PAM and its derivatives, lateral plate and intermediate mesoderm derivatives were also exposed to Cre activity, while the node, notochord, and cardiac mesoderm were not. We further demonstrate that 70-75% of the fibroblasts generated from Dll1-msd Cre, ROSA26-rtTA embryos possess Cre recombinase activity. These mice can therefore be used in combination with tet-responsive transgenic lines to generate mesoderm-derived embryonic fibroblasts that inducibly express a gene of interest.