Short-term Organ Culture Research Articles

Role of mesonephric contribution to mouse testicular development revisited

The role of the mesonephros in testicular development was re-evaluated by growing embryonic day 11.5 (E11.5) mouse testes devoid of mesonephros for 8–21 days in vivo under the renal capsule of castrated male athymic nude mice. This method provides improved growth conditions relative to previous studies based upon short-term (4–7 days) organ culture. Meticulous controls involved wholemount examination of dissected E11.5 mouse testes as well as serial sections of dissected E11.5 mouse testes which were indeed shown to be devoid of mesonephros. As expected, grafts of E11.5 mouse testes with mesonephros attached formed seminiferous tubules and also contained mesonephric derivatives. Grafts of E11.5 mouse testes without associated mesonephros also formed seminiferous tubules and never contained mesonephric derivatives. The consistent absence of mesonephric derivatives in grafts of E11.5 mouse testes grafted alone is further proof of the complete removal of the mesonephros from the E11.5 mouse testes. The testicular tissues that developed in grafts of E11.5 mouse testes alone contained canalized seminiferous tubules composed of Sox9-positive Sertoli cells as well as GENA-positive germ cells. The seminiferous tubules were surrounded by α-actin-positive myoid cells, and the interstitial space contained 3βHSD-1-positive Leydig cells. Grafts of E11.5 GFP mouse testes into wild-type hosts developed GFP-positive vasculature indicating that E11.5 mouse testes contain vascular precursors. These results indicate that the E11.5 mouse testis contains precursor cells for Sertoli cells, Leydig cells, myoid cells and vasculature whose development and differentiation are independent of cells migrating from the E11.5 mesonephros.

"Basal Cell Migration" in Regeneration of the Corneal Wound-Bed.

In this issue of Stem Cell Reports, Park et al. (2019) describe real-time in vivo visual monitoring of keratin-14+, Confetti-labeled limbal epithelial stem cells and their progeny as they contribute to central corneal wound-healing. The authors show that corneal wounds initially heal by “basal epithelial cell migration” into the wound-bed.

CLIC1 and CLIC4 complement CA125 as a diagnostic biomarker panel for all subtypes of epithelial ovarian cancer

New plasma and tissue biomarkers of epithelial ovarian cancer (EOC) could improve early diagnosis and post-diagnosis clinical management. Here we investigated tissue staining and tissue secretion of CLIC1 and CLIC4 across EOC subtypes. CLIC1 and CLIC4 are two promising biomarkers we previously showed were elevated in EOC patient sera. Individually, CLIC1 or CLIC4 stained larger percentages of malignant tumors across all EOC subtypes compared with CA125, particularly early stage and mucinous tumors. CLIC4 also stained benign tumors but staining was limited to nuclei; whereas malignant tumors showed diffuse cellular staining of stromal and tumor cells. Both proteins were shed by all EOC subtypes tumors in short term organ culture at more consistent levels than CA125, supporting their potential as pan-subtype serum and tissue biomarkers. Elevated CLIC4 expression, but not CLIC1 expression, was a negative indicator of patient survival, and CLIC4 knockdown in cultured cells decreased cell proliferation and migration indicating a potential role in tumor progression. These results suggest CLIC1 and CLIC4 are promising serum and tissue biomarkers as well as potential therapeutic targets for all EOC subtypes. This justifies development of high throughput serum/plasma biomarker assays to evaluate utility of a biomarker panel consisting of CLIC1, CLIC4 and CA125.

Development of a Short-Term Canine Full-Thickness Skin Organ Culture Method under Serum-Free Conditions

Full-thickness canine skin organ culture models could provide an entirely new opportunity for studying wound healing, keratinization disorders and allergic skin diseases, all of which have high prevalence and severe impact on canine quality of life. Here we present a canine organ culture method for the short-term maintenance of full-thickness, adult, canine skin in serum-free medium and investigate the possibility to induce mast cell degranulation ex vivo. General morphological features were maintained up to day 7. Epidermal thickness started to decrease from day 4 of culture. No changes were observed in epidermal pigmentation. Keratinocyte proliferation started to significantly decrease at day 7. Immunostaining for cytokeratin 10, cytokeratin 14 and loricrin was evident from day 1 to day 7. Compound 48/80 induced mast cell degranulation. This was the first attempt to establish a dog skin organ culture and document good preservation of most cutaneous structures till day 7. This method may help in dissecting canine skin biology in physiological and pathological conditions and to study drug mechanism of action in a biologically relevant environment.

Cone outer segment and Müller microvilli pericellular matrices provide binding domains for interphotoreceptor retinoid-binding protein (IRBP)

The close packing of vertebrate photoreceptors presents a challenge to the exchange of molecules between the outer segments, retinal pigmented epithelium (RPE), and Müller glia. An extracellular hyaluronan scaffold separates these cells while soluble interphotoreceptor matrix (IPM) proteins traffic visual cycle retinoids, fatty acids, and other molecules between them. In the IPM, retinoids and fatty acids are carried by interphotoreceptor retinoid-binding protein (IRBP). The fact that much of the retina's IRBP can be extracted by saline wash has led to the notion that IRBP does not bind to the retina, but freely distributes itself within the subretinal space. In this study, we challenge this idea by asking if there are specialized IPM domains that bind IRBP, perhaps facilitating its ability to target delivery/uptake of its ligands. Xenopus is an ideal animal model to study the role of the IPM in RPE-photoreceptor interactions. Here, we took advantage of the large size of its photoreceptors, ability to detach the retina in light, sustainability of the retina in short term organ culture, and the availability of recombinant full-length Xenopus IRBP and antisera directed against Xenopus IRBP. We compared the distribution of wash resistant native IRBP, and that of IRBP-Alexa 647 binding in Xenopus retina. IRBP and cone opsin were localized using anti-Xenopus IRBP serum, and monoclonal COS-1 respectively. Cone matrix sheath proteoglycans were localized with wheat germ agglutinin (WGA), and diffuse IPM proteoglycans with peanut agglutinin (PNA). Wholemounts and frozen sections were compared by immunofluorescence from retinas detached under Ringer's followed by additional washes, or detached directly under 4% paraformaldehyde without Ringer's wash. Undetached Lowicryl embedded retinas were subjected to IRBP immunogold electron microscopy (EM). Immunogold labeled a diffuse network of filamentous structures, and a separate distinct flocculant material directly coating the outer segments, filling the rod periciliary ridge, and associated with Müller microvilli. By immunofluorescence, Ringer's wash removed most of the diffuse IRBP, but not that coating the outer segments. IRBP-Alexa 647 bound to the cone outer segments and Müller villi region, and comparably less to rod outer segments. Co-incubation with unlabeled IRBP markedly reduced this binding; ovalbumin-Alexa 647 and Alexa 647 dye alone showed no binding. Our data suggest that the pericellular matrix of the cone outer segments and Müller microvilli provide specialized domains that facilitate IRBP's functions.

The foetal pig pineal gland is richly innervated by nerve fibres containing catecholamine-synthesizing enzymes, neuropeptide Y (NPY) and C-terminal flanking peptide of NPY, but it does not secrete melatonin.

Innervation of the mammalian pineal gland during prenatal development is poorly recognized. Therefore, immunofluorescence studies of the pineals of 70- and 90-day-old foetuses of the domestic pig were performed using antibodies against tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH), neuropeptide Y (NPY) and C-terminal flanking peptide of NPY (CPON). The investigated glands were supplied by numerous nerve fibres containing TH and DβH. The density of these fibres was higher in the distal and middle parts of the gland than in the proximal one. NPY and CPON were identified in the majority of DβH-positive fibres as well as in a small population of DβH-negative fibres localized mainly in the proximal part of the pineal. The immunoreactive fibres were more numerous in 90-day-old foetuses than in 70-day-old ones. The effect of norepinephrine on melatonin secretion by the foetal pineals in the short-term organ culture was studied to determine the role of DβH-positive fibres during prenatal life. For the same purpose melatonin was measured in the blood in the umbilical cords and in the jugular vein of the mother. The pineals of both groups of foetuses did not secrete melatonin in the organ culture, independently of the presence or absence of norepinephrine in the medium. Melatonin concentrations in the blood in the umbilical cords of foetuses from the same litter and in the jugular vein of their mother were similar. The presence of adrenergic nerve fibres in the pig pineal during gestation does not seem to be associated with the control of melatonin secretion.

Mo1065 Does Hydrotalcite Protect Esophageal Epithelium From H Ion- and Bile Acid-Induced Injury? Studies in Cultured Human Esophageal Epithelial Cells and in Short Term Organ Cultures of Rat Esophageal Tissue.

Mo1065 Does Hydrotalcite Protect Esophageal Epithelium From H Ion- and Bile Acid-Induced Injury? Studies in Cultured Human Esophageal Epithelial Cells and in Short Term Organ Cultures of Rat Esophageal Tissue.

Rapid functional upregulation of vasocontractile endothelin ETB receptors in rat coronary arteries

AimsEndothelin ETB receptors mediate under normal physiological conditions vasorelaxation in coronary arteries. However, vasocontractile ETB receptors appear in coronary arteries of ischemic heart disease patients. Interestingly, organ culture of isolated coronary arteries also induces upregulation of vasocontractile ETB receptors. This study examines the early time course and mechanism behind upregulation of contractile ETB receptors in isolated rat coronary arteries during short-term organ culture. Main methodsCoronary artery segments were mounted in wire-myographs and incubated in physiological saline solution. Contractions were measured after exposure to the specific ETB receptor agonist Sarafotoxin 6c (S6c) and the endogenous agonists endothelin-1 and endothelin-3. Protein localization and levels of ETB and phosphorylated-extracellular-signal-regulated-kinase-1/2 (ERK1/2) were examined by immunohistochemistry. Key findingsFresh arteries showed negligible vasoconstriction to S6c. However, incubation for only 4 and 7h increased S6c contractions two- and seven-fold, respectively. Furthermore, 7h incubation enhanced vasocontractile responses to endothelin-3 and increased ETB receptor density in vascular smooth muscle cells. ERK1/2 was activated rapidly after start of incubation. Moreover, incubation with either the transcriptional inhibitor actinomycin D or the mitogen-activated-protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 attenuated contractile ETB receptor upregulation. U0126 attenuated ETB receptor protein levels after 24h of incubation. SignificanceCoronary arteries rapidly upregulate vasocontractile ETB receptors during organ culture via transcriptional mechanisms and MEK-ERK1/2 signalling. This model may mimic the mechanisms seen in ischemic conditions. Furthermore, these findings have important experimental implications in tissue bath experiments lasting for more than 4h.

Herpes Simplex Virus-Induced Epithelial Damage and Susceptibility to Human Immunodeficiency Virus Type 1 Infection in Human Cervical Organ Culture

Normal human premenopausal cervical tissue has been used to derive primary cell populations and to establish ex vivo organ culture systems to study infections with herpes simplex virus (HSV-1 or HSV-2) and human immunodeficiency virus type 1 (HIV-1). Infection with either HSV-1 or HSV-2 rapidly induced multinuclear giant cell formation and widespread damage in mucosal epithelial cells. Subsequent exposure of the damaged mucosal surfaces to HIV-1 revealed frequent co-localization of HSV and HIV-1 antigens. The short-term organ culture system provides direct experimental support for the epidemiological findings that pre-existing sexually transmitted infections, including primary and recurrent herpes virus infections at mucosal surfaces, represent major risk factors for acquisition of primary HIV-1 infection. Epithelial damage in combination with pre-existing inflammation, as described here for overtly normal human premenopausal cervix, creates a highly susceptible environment for the initiation and establishment of primary HIV-1 infection in the sub-mucosa of the cervical transformation zone.

Tumor Necrosis Factor Receptor Expression and Signaling in Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC), a tubular epithelial cell (TEC) malignancy, frequently secretes tumor necrosis factor (TNF). TNF signals via two distinct receptors (TNFRs). TNFR1, expressed in normal kidney primarily on endothelial cells, activates apoptotic signaling kinase 1 and nuclear factor-kappaB (NF-kappaB) and induces cell death, whereas TNFR2, inducibly expressed on endothelial cells and on TECs by injury, activates endothelial/epithelial tyrosine kinase (Etk), which trans-activates vascular endothelial growth factor receptor 2 (VEGFR2) to promote cell proliferation. We investigated TNFR expression in clinical samples and function in short-term organ cultures of ccRCC tissue treated with wild-type TNF or specific muteins selective for TNFR1 (R1-TNF) or TNFR2 (R2-TNF). There is a significant increase in TNFR2 but not TNFR1 expression on malignant TECs that correlates with increasing malignant grade. In ccRCC organ cultures, R1-TNF increases TNFR1, activates apoptotic signaling kinase and NF-kappaB, and promotes apoptosis in malignant TECs. R2-TNF increases TNFR2, activates NF-kappaB, Etk, and VEGFR2 and increases entry into the cell cycle. Wild-type TNF induces both sets of responses. R2-TNF actions are blocked by pretreatment with a VEGFR2 kinase inhibitor. We conclude that TNF, acting through TNFR2, is an autocrine growth factor for ccRCC acting via Etk-VEGFR2 cross-talk, insights that may provide a more effective therapeutic approach to this disease.

Stability in Aromatase Immunoreactivity of Steroid-Producing Cells During Early Development of XX Gonads of the Nile tilapia, Oreochromis niloticus: an Organ Culture Study

The organ culture system is a useful tool to study the effects of various factors on the development of undifferentiated gonads. In this study, we first established an organ culture system for gonads of all genetic male and female Nile tilapia at 5-122 days after hatching (dah). This short-term (3 days) organ culture system was then used to examine the stability of the immunoreactivity of aromatase (the enzyme which converts androgen to estrogen, thus playing a crucial role in ovarian differentiation) in steroid-producing cells (SPCs). Immunohistochemical analyses revealed that aromatase-positive cells could be initially detected in the vicinity of blood vessels in the XX gonads at 7 dah. These SPCs completely lost their immunoreactivity after 3 days in culture, indicating the instability of SPCs during early ovarian differentiation. In contrast, the immunoreactivity of the SPCs was maintained to some extent even after 3 days in culture, if the gonads were from 15-23 dah. In XX gonads collected at 122 dah, there were two major populations of SPCs: one in the vicinity of the blood vessel and the other near the oocyte. The aromatase immunoreactivity was maintained in SPCs located around the oocytes, but not in those in the vicinity of the blood vessel, after 3 days in culture. These results suggest that the SPCs originate from the cells in the vicinity of the blood vessels prior to the initial ovarian differentiation in tilapia and that the degree of differentiation of SPCs is dependent on their location in the ovary.

Androgen regulation of prostate morphoregulatory gene expression: Fgf10-dependent and -independent pathways.

Androgens are essential and sufficient for prostate gland morphogenesis; however, the downstream gene targets that mediate this action are unclear. To identify androgen-regulated genes involved in prostate development, we used short-term organ culture and examined the effect of testosterone on the expression of several critical prostate morphoregulatory genes. Rat ventral prostates (VP) and lateral prostates (LP) were collected at birth, and contralateral lobes were cultured for 18 h in the presence or absence of 10 nM testosterone with or without OH-flutamide to block residual androgens. Gene expression was quantitated using real-time RT-PCR. Although expression of Fgf10, Nkx3.1, and Ptc was increased in both prostate lobes, other genes were regulated by testosterone in a lobe-specific manner. This included up-regulation of epithelial genes FgfR2iiib, Shh, Hoxb13, and Bmp7 in the VP specifically and down-regulation of mesenchymal genes Wnt5a (VP) and Bmp4 (LP). Thus, in addition to stimulation of homeobox genes and paracrine-acting growth factors, androgens may positively regulate prostatic development through suppression of growth inhibitory genes. Because previous studies revealed a similar gene regulation pattern in response to exogenous Fgf10, experiments were performed to identify androgen-regulated genes mediated through Fgf10 signaling. Short-term VP and LP cultures with FgfR antagonist PD173074 and Mek inhibitor U0126 identified epithelial Shh and Hoxb13 up-regulation by androgens to be Fgf10-dependent. We propose that androgen regulation of prostate development is mediated through positive and negative regulation of multiple morphoregulatory genes acting in combination through complex gene networks. Lobe-specific responses may provide a developmental basis for prostate gland heterogeneity.

Signal Transducer and Activator of Transcription 5b Promotes Mucosal Tolerance in Pediatric Crohn's Disease and Murine Colitis

Growth hormone (GH) regulates anabolic metabolism via activation of the STAT5b transcription factor and reduces mucosal inflammation in colitis. Peroxisome proliferator-activated receptor (PPAR) gamma suppresses mucosal inflammation and is regulated by GH through STAT5b. We hypothesized that the GH:STAT5b axis influences susceptibility to colitis via regulation of local PPARgamma abundance. Colon biopsies from children with newly diagnosed Crohn's disease (CD) and controls were exposed to GH in short-term organ culture. Trinitrobenzene sulfonic acid (TNBS) administration was used to induce colitis in STAT5b-deficient mice and wild-type controls, with and without rosiglitazone pretreatment. GH receptor, STAT5b, PPARgamma, and nuclear factor kappaB activation and expression were determined. Epithelial cell GH receptor expression and GH-dependent STAT5b activation and PPARgamma expression were reduced in CD colon. STAT5b-deficient mice exhibited reduced basal PPARgamma nuclear abundance and developed more severe proximal colitis after TNBS administration. This was associated with a significant increase in mucosal nuclear factor kappaB activation at baseline and after TNBS administration. Rosiglitazone ameliorated colitis in wild-type mice but not STAT5b-deficient mice. GH-dependent STAT5b activation is impaired in affected CD colon and contributes to chronic mucosal inflammation via down-regulation of local PPARgamma expression. Therapeutic activation of the GH:STAT5b axis therefore represents a novel target for restoring both normal anabolic metabolism and mucosal tolerance in CD.

Prediction of Eye Irritation Potential of Surfactant-Based Rinse-Off Personal Care Formulations by the Bovine Corneal Opacity and Permeability (BCOP) Assay

Evaluation of eye irritation potential is a routine part of product safety testing. Many companies have elected to use in vitro methods for this evaluation. We used the bovine corneal opacity and permeability (BCOP) assay for the prediction of the eye irritation potential of surfactant-based rinse-off personal care formulations in this study because of its positive performance in previous studies (), and its potential to measure depth of injury through histological evaluation. The BCOP uses isolated corneas maintained in short-term organ culture and measures changes in opacity and epithelial barrier integrity (as determined by fluorescein passage through the cornea). Surfactants (anionic and nonionic) used in personal care formulations induce only small increases in direct opacity but the degree of epithelial damage is reflected in permeability measurement (OD490). The permeability measurement is the primary endpoint for this class of products. Marketed liquid hand soap was selected as the benchmark due to extensive in vivo and market history data. Using the liquid hand soap, a 25% v/v aqueous dilution with a 30-minute exposure produced the optimal resolution. A series of products with known in vivo eye irritation scores were evaluated to establish a prediction model. Histological evaluation was used to compare the degree of tissue damage to the permeability scores. Based on the permeability scores and histological review, the testing protocol for a surfactant-based rinse-off personal care formulation was developed using a 25% v/v aqueous dilution, a 30-minute exposure, concurrent testing of the benchmark control, and use of permeability measurements as the endpoint for the evaluation of eye irritation potential.

Analyzing checkpoint controls in human skin.

A short-term whole-skin organ culture model has been established to enable the investigation of cell cycle perturbations in epidermal layer cells following exposure to ultraviolet radiation (UVR). This model affords the opportunity to manipulate the growth and nutrient conditions, and to perform detailed biochemical and immunohistochemical analysis of skin cells in their normal epidermal layer microenvironment. The use of this model is described in this chapter.

Arginase 1 Overexpression in Psoriasis: Limitation of Inducible Nitric Oxide Synthase Activity as a Molecular Mechanism for Keratinocyte Hyperproliferation

Abnormal proliferation of keratinocytes in the skin appears crucial to the pathogenesis of psoriasis, but the underlying mechanisms remain unknown. Nitric oxide (NO), released from keratinocytes at high concentrations, is considered a key inhibitor of cellular proliferation and inducer of differentiation in vitro. Although high-output NO synthesis is suggested by the expression of inducible NO synthase (iNOS) mRNA and protein in psoriasis lesions, the pronounced hyperproliferation of psoriatic keratinocytes may indicate that iNOS activity is too low to effectively deliver anti-proliferative NO concentrations. Here we show that arginase 1 (ARG1), which substantially participates in the regulation of iNOS activity by competing for the common substrate L-arginine, is highly overexpressed in the hyperproliferative psoriatic epidermis and is co-expressed with iNOS. Expression of L-arginine transporter molecules is found to be normal. Treatment of primary cultured keratinocytes with Th1-cytokines, as present in a psoriatic environment, leads to de novo expression of iNOS but concomitantly a significant down-regulation of ARG1. Persistent ARG1 overexpression in psoriasis lesions, therefore, may represent a disease-associated deviation from normal expression patterns. Furthermore, the culturing of activated keratinocytes in the presence of an ARG inhibitor results in a twofold increase in nitrite accumulation providing evidence for an L-arginine substrate competition in human keratinocytes. High-output NO synthesis is indeed associated with a significant decrease in cellular proliferation as shown by down-regulation of Ki67 expression in cultured keratinocytes but also in short-term organ cultures of normal human skin. In summary, our data demonstrate for the first time a link between a human inflammatory skin disease, limited iNOS activity, and ARG1 overexpression. This link may have substantial implications for the pathophysiology of psoriasis and the development of new treatment strategies.

Experimental endometriosis: the nude mouse as a xenographic host.

Endometriosis is a complex disease that can develop as a consequence of retrograde menstruation, occurring in association with the cyclic loss of endometrial tissue in primates and humans. In addition, progression of disease parallels a woman's exposure to ovarian steroids, rarely occurring prior to menarche and generally resolving following menopause. Because of the cost of developing primate models to study endometriosis, numerous small animal models have been established to approach various elements related to the pathophysiology of this disease. Our laboratory has developed an experimental endometriosis model using nude mice as a xenographic host for human tissues. Our goal is to approach the basic cellular mechanisms of estrogen and progesterone action that link these hormones to the development or prevention of endometriosis. In our initial studies, we have sought to understand steroid-associated regulation of matrix metalloproteinases (MMPs) with regard to the development of experimental endometriosis. Using both short-term organ cultures and nude mice as xenographic hosts of human tissue, we have demonstrated a critical role of progesterone and progesterone-associated cytokines in preventing the initial establishment of experimental disease. Women with endometriosis appear to lack normal endometrial responsiveness to progesterone, resulting in altered expression of several MMPs and an enhanced ability of these tissues to establish ectopic lesions in nude mice. Developing a better understanding of the impairments in the normal endometrial physiology of women with endometriosis should aid in the development of better treatment or diagnostic strategies.

Ultraviolet A1 Radiation Induces Nitric Oxide Synthase-2 Expression in Human Skin Endothelial Cells in the Absence of Proinflammatory Cytokines

Skin exposure to ultraviolet radiation from sunlight causes erythema and edema formation as well as inflammatory responses. As some of these ultraviolet-induced effects are potentially mediated by nitric oxide synthases, we examined the role of cytokines and ultraviolet A1 radiation (340-400 nm) on the expression of the nitric oxide synthase-2 in endothelia of normal human skin biopsies during short-term organ culture as well as expression and activity of the nitric oxide synthase-2 in in vitro cell cultures of human dermal endothelial cells. Both, cytokine challenge (interleukin-1beta + tumor necrosis factor-alpha + interferon-gamma) but also ultraviolet A1 exposure (50 J per cm2) in the absence of cytokines led to the expression of nitric oxide synthase-2 in human skin organ cultures as shown by immunohistochemistry. Moreover, exposing human dermal endothelial cell cultures to proinflammatory cytokines but also to ultraviolet A1 radiation (6-24 J per cm2) in the absence of cytokines resulted in significant nitric oxide synthase-2 mRNA and protein expression as well as enzyme activity. Ultraviolet A1 irradiation of cytokine activated cells led to further increases in nitric oxide synthase-2 mRNA, protein expression, and enzyme activity. Moreover, a reporter gene assay using a human nitric oxide synthase-2 promoter construct provide evidence that ultraviolet A1, in the absence of cytokines, induces nitric oxide synthase-2 expression and activity, as previously shown for cytokines. Thus, the results presented here demonstrate for the first time that in dermal endothelia of human skin ultraviolet A1 radiation alone represents a proinflammatory stimulus sufficient to initiate nitric oxide synthase-2 expression as well as activity comparable with the respective response seen in the presence of proinflammatory cytokines.

Leukaemia Inhibitory Factor (LIF) Expression in Skin from Amyotrophic Lateral Sclerosis Patients Compared with Skin from Normal Individuals: What is the Function of LIF in the Skin?

Leukaemia Inhibitory Factor (LIF) Expression in Skin from Amyotrophic Lateral Sclerosis Patients Compared with Skin from Normal Individuals: What is the Function of LIF in the Skin?

Selective expression of the proprotein convertases furin, pc5, and pc7 in proliferating vascular smooth muscle cells of the rat aorta in vitro.

The aim of this study was to investigate whether transformation of quiescent vascular smooth muscle cells (VSMCs) into proliferating secretory cells is accompanied by an expression of processing enzymes that activate de novo-synthesized growth factors. Three enzymes belonging to the family of the kexin/subtilisin-like mammalian proprotein convertases (PCs), furin, PC5, and PC7, were found to be upregulated after balloon denudation in vivo. To determine their importance in these cell processes, we investigated their gene regulation using a short-term organ culture system. After incubation of rat aorta for 4 and 24 hr in serum-free medium, we demonstrated a significant induction of VSMC proliferation. The affected subset of VSMCs, positive for alpha-smooth muscle actin, also expressed proliferating cell nuclear antigen (PCNA). Our results revealed a parallel upregulation of furin, PC5, and PC7 in PCNA-immunolabeled cells. As a substrate model for comparison with PCs we used nerve growth factor (NGF). NGF is known to be activated by PCs. As shown by Northern blotting analysis, NGF mRNA concentration was significantly increased in cultured explants. NGF was released into the culture medium. In conclusion, both PCs and NGF are coordinately modulated on induction of VSMC proliferation.