Basement Membrane Protein Laminin Research Articles

TGFβ1 Regulates Cellular Composition of In Vitro Cardiac Perivascular Niche Based on Cardiospheres.

The cardiac perivascular niche is a cellular microenvironment of a blood vessel. The principles of niche regulation are still poorly understood. We studied the effect of TGFβ1 on cells forming the cardiac perivascular niche using 3D cell culture (cardiospheres). Cardiospheres contained progenitor (c-Kit), endothelial (CD31), and mural (αSMA) cells, basement membrane proteins (laminin) and extracellular matrix proteins (collagen I, fibronectin). TGFβ1 treatment decreased the length of CD31+ microvasculature, VE cadherin protein level, and proportion of NG2+ cells, and increased proportion of αSMA+ cells and transgelin/SM22α protein level. We supposed that this effect is related to the stabilizing function of TGFβ1 on vascular cells: decreased endothelial cell proliferation, as shown for HUVEC, and activation of mural cell differentiation.

O20 Dissecting junctional epidermolysis bullosa heterogeneity

Abstract Introduction and aims Epidermolysis bullosa (EB) is a family of rare, incurable, inherited disorders that are characterized by extreme skin and mucosal fragility. One of the most severe forms is junctional EB (JEB). JEB is caused by mutations in genes encoding the skin basement membrane proteins laminin 332 (α3, β3, γ2), type XVII collagen or integrin α6β4. Despite similarities between the disease pathology of JEB and other forms of EB there is a dramatic reduction in life expectancy for patients with JEB. Patients with JEB rarely survive beyond the first year of life. To investigate this disparity, we have optimized a previously published conditional tamoxifen induced knockout JEB mouse model (Lama3flx/flxK14CreERT). This mouse model recapitulates all key features of JEB, such as thickening of the epidermis and visible blistering owing to the loss of laminin α3 at the basement membrane. Methods Single-cell (sc)RNA-Seq analysis was conducted on pooled skin samples from a healthy (control) and JEB mouse model (n = 3 per genotype). Immunofluorescent staining was conducted to confirm scRNA-Seq analysis including markers for skin differentiation (K14, transglutaminase 1, loricrin), proliferation (Ki67), basement membrane (laminin α3, laminin β3, laminin γ2, collagen VII) and immune markers (F4/80, Ly6-G/C, CD3, CD79). Results We can see large differences in cellular populations across the two conditions. With increased T- and B-immune-cell subpopulations in the JEB model compared with the healthy control. Utilizing CellChatDB we identified disease-specific signalling pathways that identify activated T- and B-cell populations interacting with JEB macrophage populations via proinflammatory pathways. Conclusions We have utilized an in vivo mouse model to gain insight into the molecular and cellular differences in a JEB diseased phenotype. Identifying changes in cellular pathways could be used as a targeted approach to identify a beneficial therapeutic treatment for JEB.

O19 Improvement of a junctional epidermolysis bullosa mouse model

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is the most severe of the epidermolysis bullosa (EB) disorders and is caused by mutations in genes encoding the skin basement membrane proteins laminin 332, type XVII collagen or integrin α6β4. Affected individuals experience blistering from birth leading to scarring, granulation tissue and susceptibility to infection. In JEB, generalized severe, there is 50% mortality in the first 2 years of life, often owing to failure to thrive or overwhelming infection. A number of animal models for JEB exist; however, because of severe blistering on the entire surface of the skin, these mice die soon after birth, reducing their experimental utility. In 2017, the first adult inducible knockout mouse model of JEB was published. In this mouse model, the disruption of the Lama3 gene is specifically induced in epidermal basal keratinocytes after birth. These mice remain viable, which allows us to study the longer-term consequences of the loss of laminin α3. However, the mice did not develop the severe phenotype observed in patients with JEB. Methods We have optimized this previously published methodology of a conditional knockout JEB mouse model (Lama3flx/flxK14CreERT) to now include more regular tamoxifen injections, which Results in a more controlled and severe phenotype of JEB. Results Widespread skin blistering develops after 12 weeks of tamoxifen treatment with an impaired skin barrier (transepidermal water loss) and a loss of epidermal lipids. Immunofluorescent staining was conducted to assess skin differentiation (K14, transglutaminase 1, loricrin), proliferation (Ki67), basement membrane proteins (laminin α3, laminin β3, laminin γ2, collagen VII) and immune markers (F4/80, Ly6-G/C, CD3, CD79). Conclusions These mice recapitulate all key features of human JEB with loss of laminin α3 at the basement membrane, epidermal thickening and visible blisters. This allows us to delve deeper into the functional and downstream pathways affected by the loss of laminin α3 and discover why the prognosis of patients with JEB is so severe.

Distinct uptake and elimination profiles for trastuzumab, human IgG, and biocytin-TMR in experimental HER2+ brain metastases of breast cancer.

The aim of this study is an improved understanding of drug distribution in brain metastases. Rather than single point snapshots, we analyzed the time course and route of drug/probe elimination (clearance), focusing on the intramural periarterial drainage (IPAD) pathway. Mice with JIMT1-BR HER2+ experimental brain metastases were injected with biocytin-TMR and either trastuzumab or human IgG. Drugs/probes circulated for 5 min to 48 h, followed by perfusion. Brain sections were stained for human IgG, vascular basement membrane proteins laminin or collagen IV, and periarterial α-SMA. A machine learning algorithm was developed to identify metastases, metastatic microenvironment, and uninvolved brain in confocally scanned brain sections. Drug/probe intensity over time and total imaged drug exposure (iAUC) were calculated for 27,249 lesions and co-immunofluorescence with IPAD-vascular matrix analyzed in 11,668 metastases. In metastases, peak trastuzumab levels were 5-fold higher than human IgG but 4-fold less than biocytin-TMR. The elimination phase constituted 85-93% of total iAUC for all drugs/probes tested. For trastuzumab, total iAUC during uptake was similar to the small molecule drug probe biocytin-TMR, but slower trastuzumab elimination resulted in a 1.7-fold higher total iAUC. During elimination trastuzumab and IgG were preferentially enriched in the α-SMA+ periarterial vascular matrix, consistent with the IPAD clearance route; biocytin-TMR showed heterogeneous elimination pathways. Drug/probe elimination is an important component of drug development for brain metastases. We identified a prolonged elimination pathway for systemically administered antibodies through the periarterial vascular matrix that may contribute to the sustained presence and efficacy of large antibody therapeutics.

Increasing reproducibility in preclinical stroke research: the correlation of immunofluorescence intensity measurements and Western blot analyses strongly depends on antibody clonality and tissue pre-treatment in a mouse model of focal cerebral ischemia.

In the setting of stroke, ischemia not only impairs neuronal function, but also detrimentally affects the different components of the neurovascular unit, which are shown to be involved in the transition from reversible to long-lasting tissue damage. In this context, the glial proteins myelin basic protein (MBP) and the 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) as well as the vasculature-associated basement membrane proteins laminin and collagen IV have been identified as ischemia-sensitive elements. However, available data from immunofluorescence and Western blot analyses are often found to be contradictory, which renders interpretation of the respective data rather difficult. Therefore, the present study investigates the impact of tissue pre-treatment and antibody clonality on immunofluorescence measurements of the mentioned proteins in a highly reproducible model of permanent middle cerebral artery occlusion. Here, immunofluorescence labeling using polyclonal antibodies revealed an increased immunofluorescence intensity of MBP, CNP, laminin and collagen IV in ischemic areas, although Western blot analyses did not reveal increased protein levels. Importantly, contrary to polyclonal antibodies, monoclonal ones did not provide increased fluorescence intensities in ischemic areas. Further, we were able to demonstrate that different ways of tissue pre-treatment including paraformaldehyde fixation and antigen retrieval may not only impact on fluorescence intensity measurements in general, but rather one-sidedly affect either ischemic or unaffected tissue. Therefore, immunofluorescence intensity measurements do not necessarily correlate with the actual protein levels, especially in ischemia-affected tissue and should always be complemented by different techniques to enhance reproducibility and to hopefully overcome the translational roadblock from bench to bedside.

Fibronectin‐Mediated Inflammatory Signaling Through Integrin α5 in Vascular Remodeling

BackgroundAdhesion of vascular endothelial cells to the underlying basement membrane potently modulates endothelial cells to cells' inflammatory activation. The normal basement membrane proteins laminin and collagen IV attenuate inflammatory signaling in part through integrin α2β1. In contrast, fibronectin, the provisional matrix protein found in injured, remodeling or inflamed vessels, sensitizes endothelial cells to inflammatory stimuli through integrins α5β1and and αvβ3. A chimeric integrin in which the cytoplasmic domain of α5 is replaced with that of α2 pairs with β1 and binds fibronectin but signals like α2β1.Methods and ResultsHere, we examined mice in which integrin α5 is replaced with the α5/2 chimera, using the transverse aortic constriction and partial carotid ligation models of vessel remodeling. Following transverse aortic constriction and partial carotid ligation surgery, wild‐type mice showed increased fibronectin deposition and expression of inflammatory markers, which were strongly attenuated in a5/2 mice. α5/2 mice also showed reduced artery wall hypertrophy in the transverse aortic constriction model and diminished inward remodeling in the partial carotid ligation model. Acute atherosclerosis after partial carotid ligation in hyperlipidemic ApoE−/− mice on a high fat diet was dramatically decreased in α5/2 mice.ConclusionsFibronectin and integrin α5 signaling is a key element of pathological vascular remodeling in acute models of both hypertension and disturbed flow. These results underscore the key role for integrin α5 signaling in pathological vascular remodeling associated with hypertension and atherosclerosis and support its potential as a therapeutic target.

Cytokeratin 5 determines maturation of the mammary myoepithelium

SummaryAt invasion, transformed mammary epithelial cells expand into the stroma through a disrupted myoepithelial (ME) cell layer and basement membrane (BM). The intact ME cell layer has thus been suggested to act as a barrier against invasion. Here, we investigate the mechanisms behind the disruption of ME cell layer. We show that the expression of basal/ME proteins CK5, CK14, and α-SMA altered along increasing grade of malignancy, and their loss affected the maintenance of organotypic 3D mammary architecture. Furthermore, our data suggests that loss of CK5 prior to invasive stage causes decreased levels of Zinc finger protein SNAI2 (SLUG), a key regulator of the mammary epithelial cell lineage determination. Consequently, a differentiation bias toward luminal epithelial cell type was detected with loss of mature, α-SMA-expressing ME cells and reduced deposition of basement membrane protein laminin-5. Therefore, our data discloses the central role of CK5 in mammary epithelial differentiation and maintenance of normal ME layer.

Loss of the laminin subunit alpha-3 induces cell invasion and macrophage infiltration in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood. To study the effect that loss of the laminin subunit alpha-3 (α3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment. We examined the role of the basement membrane proteins laminin subunits α3, β3 and γ2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA-Seq, mouse xenograft models and patient tumour samples. Analysis of SCC tumours and cell lines using antibodies specific to laminin chains α3, β3 and γ2 identified a link between poorly differentiated SCC and reduced expression of laminin α3 but not the other laminin subunits investigated. Knockdown of laminin α3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin α3. Inhibition of ROCK (rho-associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin α3 knockdown cells. Knockdown of laminin subunits α3 and γ2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin α3 correlated with increased tumour-associated macrophages both in xenografted tumours and in patient tumour samples. These data provide evidence that loss of the laminin α3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment.

PATH-04. THE BLOOD-BRAIN BARRIER IN DIFFUSE MIDLINE GLIOMA AND ITS IMPLICATIONS FOR DRUG DELIVERY

Abstract INTRODUCTION Chemotherapy has been unsuccessful for pediatric diffuse midline glioma (DMG) most likely due to an intact blood-brain barrier (BBB). However, the BBB has not been characterized in DMG and therefore its implications for drug delivery are unknown. In this study we characterized the BBB in DMG patients and compared this to healthy controls. METHODS End-stage DMG pontine samples (n=5) were obtained from the VUmc diffuse intrinsic pontine glioma (DIPG) autopsy study and age-matched healthy pontine samples (n=22) were obtained from the NIH NeuroBioBank. Tissues were stained for BBB markers claudin-5, zonula occludens-1, laminin, and PDGFRβ. Claudin-5 stains were used to determine vascular density and diameter. RESULTS In DMG, expression of claudin-5 was reduced and dislocated to the abluminal side of endothelial cells. In addition, the expression of zonula occludens-1 was reduced. The basement membrane protein laminin expression was reduced at the glia limitans in both pre-existent vessels and neovascular proliferation. PDGFRβ expression was not observed in DMG but was present in healthy pons. Furthermore, the number of blood vessels in DMG was significantly (P< 0.01) reduced (13.9 ± 11.8/mm2) compared to healthy pons (26.3 ± 14.2/mm2). Markedly, the number of small blood vessels (< 10µm) was significantly lower (P< 0.01) while larger blood vessels (> 10µm) were not significantly different (P= 0.223). The mean vascular diameter was larger for DMG 9.3 ± 9.9µm compared to 7.7 ± 9.0µm for healthy pons (P= 0.016). CONCLUSION Both the BBB and the vasculature are altered at end-stage DMG. The reduced vascular density might have implications for several drug delivery methods such as focused ultrasound and convection enhanced delivery that are being explored for the treatment of DMG. The functional effects of the structurally altered BBB remain unknown and further research is needed to evaluate the BBB integrity at end-stage DMG

Abstract 346: Recognition of anoikis resistant subpopulation in colorectal carcinoma and their association to adverse prognosis

Abstract Background: Colorectal cancer is the third most common cause of death in western countries. Anoikis, is a form of cell death induced by the loss of the contact to extracellular matrix. Resistance to anoikis has been reported to enable metastatic and invasive potential in malignant tumors. Therefore cell populations with anoikis resistance ability are crucial to be identified and targeted and yet so far only studied with anti-adhesion tests in vitro. Hence, we aimed at identifying and quantifying cell populations with anoikis resistance ability. Identification of these cell populations was based on recognition of cells with absence of both contact with the extracellular matrix and evidence of increased apoptosis rate. Materials and methods: We used TMA material from a well characterized series of colorectal cancer patients (n=137) for identification and quantification of putative anoikis resistant (AR) subpopulations. We stained the specimens with hematoxylin and eosin and performed immunohistochemical stainings with antibodies to Ki-67 for proliferation rate, cleaved cytokeratin 18 (M30) for apoptosis rate, and for basement membrane (BM) proteins laminin and collagen IV. Carcinoma cell subpopulations with features of anoikis resistance were quantitated by using digital image analysis and their correlation with clinicopathological features and prognostic effect were analyzed. Results: We identified micropapillary structures (MIPs;) presenting as focal piles of epithelial cells in columnar epithelium, cribriform and solid structures formed by carcinoma cells to be the subpopulations containing cells without ECM contact. All these three putative AR subpopulations showed lower apoptosis index as compared with other carcinoma cells. MIPs also presented with low PI. No clear association with clinicopathological features or KRAS or BRAF mutations were seen. Abundance of AR subpopulations was an independent prognostic factor and associated with a significantly worse patient survival. Conclusion: Lower apoptosis rate in all three putative AR structures along with absence of basement membrane protein contact indicates ability of these cells to survive without matrix contact, i.e. anoikis is inhibited in them. Association of low apoptosis and proliferation rates in MIPs indicates this subpopulation being distinct to other putative AR subpopulations in cribriform and solid structures, and likely to represent a quiescent population. Our histopathological assay of anoikis resistance provides new prognostic information. More studies are needed to confirm these findings, to disclose the role of specific mutations in their pathogenesis, and to analyze whether their presence predicts responses to treatment modalities. Citation Format: Madhura Patankar. Recognition of anoikis resistant subpopulation in colorectal carcinoma and their association to adverse prognosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 346.

Draxin alters laminin organization during basement membrane remodeling to control cranial neural crest EMT

Premigratory neural crest cells arise within the dorsal neural tube and subsequently undergo an epithelial-to-mesenchymal transition (EMT) to leave the neuroepithelium and initiate migration. Draxin is a Wnt modulator that has been shown to control the timing of cranial neural crest EMT. Here we show that this process is accompanied by three stages of remodeling of the basement membrane protein laminin, from regression to expansion and channel formation. Loss of Draxin results in blocking laminin remodeling at the regression stage, whereas ectopic maintenance of Draxin blocks remodeling at the expansion stage. The latter effect is rescued by addition of Snail2, previously shown to be downstream of Draxin. Our results demonstrate an essential function for the Wnt modulator Draxin in regulating basement membrane remodeling during cranial neural crest EMT.

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix.

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17%±7.5%) and higher postpurification islet recovery (83.8%±5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

Development of Self-Assembled Nanoribbon Bound Peptide-Polyaniline Composite Scaffolds and Their Interactions with Neural Cortical Cells.

Degenerative neurological disorders and traumatic brain injuries cause significant damage to quality of life and often impact survival. As a result, novel treatments are necessary that can allow for the regeneration of neural tissue. In this work, a new biomimetic scaffold was designed with potential for applications in neural tissue regeneration. To develop the scaffold, we first prepared a new bolaamphiphile that was capable of undergoing self-assembly into nanoribbons at pH 7. Those nanoribbons were then utilized as templates for conjugation with specific proteins known to play a critical role in neural tissue growth. The template (Ile-TMG-Ile) was prepared by conjugating tetramethyleneglutaric acid with isoleucine and the ability of the bolaamphiphile to self-assemble was probed at a pH range of 4 through 9. The nanoribbons formed under neutral conditions were then functionalized step-wise with the basement membrane protein laminin, the neurotropic factor artemin and Type IV collagen. The conductive polymer polyaniline (PANI) was then incorporated through electrostatic and π–π stacking interactions to the scaffold to impart electrical properties. Distinct morphology changes were observed upon conjugation with each layer, which was also accompanied by an increase in Young’s Modulus as well as surface roughness. The Young’s Modulus of the dried PANI-bound biocomposite scaffolds was found to be 5.5 GPa, indicating the mechanical strength of the scaffold. Thermal phase changes studied indicated broad endothermic peaks upon incorporation of the proteins which were diminished upon binding with PANI. The scaffolds also exhibited in vitro biodegradable behavior over a period of three weeks. Furthermore, we observed cell proliferation and short neurite outgrowths in the presence of rat neural cortical cells, confirming that the scaffolds may be applicable in neural tissue regeneration. The electrochemical properties of the scaffolds were also studied by generating I-V curves by conducting cyclic voltammetry. Thus, we have developed a new biomimetic composite scaffold that may have potential applications in neural tissue regeneration.

Performance of conventional histochemical methods relative to a novel immunolabeling technique in assessing degree of degradation in comminuted chicken meat

The potential of a novel immunohistochemical method detecting the muscle protein meromyosin and the basement membrane protein laminin to objectively and accurately determine the degree of degradation of muscle tissue in comminuted chicken meat was investigated in comparison to three different conventional methods, two slightly different toluidine blue (TB) staining procedures and a haematoxylin-eosin (HE) staining. All samples were evaluated for degree of muscle degradation, one method by image analysis system and three by visual assessment of degree of degradation. Analyses were carried out on reference samples consisting of mixtures of emulsified and minced meat in steps of 10%. The immunohistochemical approach (LAM) showed better selectivity and good image quality. The correlation (R) between the amount of minced meat in mixtures and degree of degradation were higher and consistent by this approach (0.94 compared with 0.62, 0.61, and 0.85 for thigh muscles by LAM, HE, TB-Max Rubner-Institute (MRI) and TB-Leatherhead Food Research (LFR), respectively, and 0.91 compared with 0.73, 0.73 and 0.98 for breast muscle by LAM, HE, TB-MRI and TB-LFR, respectively). When applied to industrial samples, the results showed that it is possible by all methods to separate at least two different groups based on degree of degradation. In conclusion, the good image contrast, specificity of the staining and objectivity and transparency of the measurement speaks in favor of the LAM method as optimal to quantify degree of degradation of muscle structure in comminuted and fragmented chicken meat and obviously a likely candidate for a standard method.

Requirement for basement membrane laminin α5 during urethral and external genital development

Hypospadias, a congenital malformation of the penis characteristic of an abnormal urethral orifice, affects 1 in every 125 boys, and its incidence is rising. Herein we test the hypothesis that the basement membrane protein laminin α5 (LAMA5) plays a key role in the development of the mouse genital tubercle, the embryonic anlage of the external genitalia. Using standard histological analyses and electron microscopy, we characterized the morphology of the external genitalia in Lama5 knockout (LAMA5-KO) mouse embryos during both androgen-independent genital tubercle development and androgen-mediated sexual differentiation. We compared regulatory gene expression between control and LAMA5-KO by in situ hybridization. We also examined the epithelial structure of the mutant genital tubercle using immunofluorescence staining and histological analyses of semi-thin sections. We found that Lama5 was expressed in both ectodermal and endodermal epithelia of the cloaca. The LAMA5-KO displayed a profound external genital malformation in which the genital tubercle was underdeveloped with a large ectopic orifice at the proximal end. In older embryos, the urethra failed to form a tubular structure and was left completely exposed. These defects were not associated with a significant alteration in regulatory gene expression, but rather with a defective ectodermal epithelium and an abnormal disintegration of the cloacal membrane. We conclude that LAMA5 is required in the basement membrane to maintain normal architecture of the ventral ectoderm during genital tubercle development, which is essential for the formation of a tubular urethra. Perturbation of LAMA5, and possibly other basement membrane components, may cause hypospadias in humans.

Biofunctionalization of PEDOT films with laminin-derived peptides

Poly(3,4-ethylenedioxythiophenes) (PEDOT) have been extensively explored as materials for biomedical implants such as biosensors, tissue engineering scaffolds and microelectronic devices. Considerable effort has been made to incorporate biologically active molecules into the conducting polymer films in order to improve their long term performance at the soft tissue interface of devices, and the development of functionalized conducting polymers that can be modified with biomolecules would offer important options for device improvement. Here we report surface modification, via straightforward protocols, of carboxylic-acid-functional PEDOT copolymer films with the nonapeptide, CDPGYIGSR, derived from the basement membrane protein laminin. Evaluation of the modified surfaces via XPS and toluidine blue O assay confirmed the presence of the peptide on the surface and electrochemical analysis demonstrated unaltered properties of the peptide-modified films. The efficacy of the peptide, along with the impact of a spacer molecule, for cell adhesion and differentiation was tested in cell culture assays employing the rat pheochromocytoma (PC12) cell line. Peptide-modified films comprising the longest poly(ethylene glycol) (PEG) spacer used in this study, a PEG with ten ethylene glycol repeats, demonstrated the best attachment and neurite outgrowth compared to films with peptides alone or those with a PEG spacer comprising three ethylene glycol units. The films with PEG10-CDPGYISGR covalently modified to the surface demonstrated 11.5% neurite expression with a mean neurite length of 90μm. This peptide immobilization technique provides an effective approach to biofunctionalize conducting polymer films. Statement of SignificanceFor enhanced diagnosis and treatment, electronic devices that interface with living tissue with minimum shortcomings are critical. Towards these ends, conducting polymers have proven to be excellent materials for electrode-tissue interface for a variety of biomedical devices ranging from deep brain stimulators, cochlear implants, and microfabricated cortical electrodes. To improve the electrode-tissue interface, one strategy utilized by many researchers is incorporating relevant biological molecules within or on the conducting polymer thin films to provide a surface for cell attachment and/or provide biological cues for cell growth. The present study provides a facile means for generating PEDOT films grafted with a laminin peptide with or without a spacer molecule for enhanced cell attachment and neurite extension.

Fetal Development of the Bovine Uterus: A Light Microscopy and Immunohistochemical Study

Important steps during the prenatal development of the bovine uterus are described using conventional hematoxylin-eosin staining of fetuses from different developmental stages [crown-rump length (CRL) 9.2-94.0 cm]. Additionally, a number of intermediate filaments (keratin 7, 8, 14, 18, 19; and vimentin), the basement membrane protein laminin, smooth-muscle marker (SMA), and S100 were studied to further characterize certain differentiation processes. During early development, the uterine epithelium is simple or (pseudo)stratified with bud-like protrusions. Developing caruncles can be observed in the corpus uteri at a CRL of 15.8 cm onwards, showing a simple, keratin-positive epithelium. In contrast, the intercaruncular areas are characterized by a (pseudo)stratified epithelium, which also shows positive staining in a different manner for the investigated keratins. A differentiation of smooth muscle cell layers can be observed from a CRL of 24.4 cm onwards. Intense SMA-positive cells/fibers, arranged perpendicularly to the developing circular SMA-positive muscle cell layer, can be found preferentially located in the developing caruncles. Lymphocytes occur in the uterine epithelium and stroma in the corpora and cornua of fetuses with a CLR of 15.8 cm and higher.

Ex Vivo Prefabricated Rat Skin Flap Using Cell Sheets and an Arteriovenous Vascular Bundle

Recently, research on tissue-engineered skin substitutes have been active in plastic surgery, and significant development has been made in this area over the past several decades. However, a regenerative skin flap has not been developed that could provide immediate blood flow after transplantation. Here, we make a regenerative skin flap ex vivo that is potentially suitable for microsurgical transplantation in future clinical applications. In rats, for preparing a stable vascular carrier, a femoral vascular pedicle was sandwiched between collagen sponges and inserted into a porous chamber in the abdomen. The vascular bed was harvested 3 weeks later, and extracorporeal perfusion was performed. A green fluorescent protein positive epidermal cell sheet was placed on the vascular bed. After perfusion culture, the whole construct was harvested and fixed for morphological analyses. After approximately 10 days perfusion, the epidermal cell sheet cornified sufficiently. The desquamated corneum was positive for filaggrin. The basement membrane protein laminin 332 and type 4 collagen were deposited on the interface area between the vascular bed and the epidermal cell sheet. Moreover, an electron microscopic image showed anchoring junctions and keratohyalin granules. These results show that we were able to produce native-like skin. We have succeeded in creating regenerative skin flap ex vivo that is similar to native skin, and this technique could be applied to create various tissues in the future.

Nephrology Crossword: Genetics and glomerulopathies

ACROSS 3. Monoclonal antibody used in the treatment of atypical hemolytic uremic syndrome, a disorder caused by mutations in regulatory components of complement system. 5. Protein originally identified in lymphocytes but also critical in slit diaphragm structure and assembly; mutations in encoding gene result in proteinuric renal disease and foot process effacement (abbv). 7. Syndrome of isolated glomerular hematuria caused by mutations in type IV collagen (abbv). 9. First ion channel to be implicated in hereditary nephrotic syndrome and focal segmental glomerulosclerosis (FSGS); mutation results in increased calcium influx, which disrupts podocyte structure and function (abbv). 10. Gene critical for normal embryonic development of the urogenital system and kidneys; mutation results in nephrotic syndrome, urogenital anomalies, and predisposition to urogenital tumors (abbv). 11. Transmembrane protein critical to slit diaphragm structure and podocyte function; mutations in encoding gene cause congenital nephrotic syndrome of Finnish type. 15. Distinct clinical syndrome of hematuria, proteinuria, sensorineural deafness, and ocular abnormality caused by mutations in type 4 collagen family. 16. Gene encoding glomerular basement membrane (GBM) protein laminin b2; mutation results in clinical syndrome of ocular defects (microcoria), neurological abnormalities, and mesangial sclerosis with onset shortly after birth (abbv). 17. Gene identified to confer additional risk for FSGS and glomerulosclerosis from secondary causes in African Americans (abbv). DOWN 1. Mutation in encoded protein __ results in dysregulation of cross-linking of actin filaments in podocyte foot processes and presents as FSGS. 2. Mutation in the encoding gene results in __ enzyme deficiency and accumulation of glycol-sphingolipids, presents as renal failure in the third to sixth decade. 4. Gene critical in dorsoventral patterning of the vertebrae limb; mutations cause nail patella syndrome (abbv). 6. Recently identified mutation that results in hemolytic uremic syndrome without complement activation, suggesting alternate pathophysiological mechanisms1 (abbv). 8. __ deficiency causes abnormalities of lipid metabolism and proteinuric kidney disease with foamy appearance of GBM secondary to lipid deposition (abbv). 12. Hairpin-shaped protein of podocytes; mutation results in malfunction of slit diaphragm and steroid-resistant nephrotic syndrome in children. 13. Mutation in this gene results in defective podocyte development, diffuse mesangial sclerosis, and infantile nephrotic syndrome (abbv). 14. Mutation in this formin gene causes mislocalization of encoded protein, resulting in FSGS in early adolescence to adulthood (abbv). http://www.kidney-international.org m a k e y o u r d i a g n o s i s

A2E‐Mediated Photochemical Modification to Fibronectin and its Implications to Age‐Related Changes in Bruch's Membrane

Lipofuscin accumulates normally with age and is more pronounced in retinal dystrophies such as age-related macular degeneration. The major bis-retinoid component of lipofuscin is A2E. In addition to cell damage effects by A2E, we have previously demonstrated that blue-light-mediated A2E leads to modifications in the basement membrane protein laminin. Therefore, the purpose of this study was to advance the understanding of A2E photooxidation effects on fibronectin, the major glycoprotein of Bruch's membrane. In this study, A2E was irradiated with blue light in the presence of a fibronectin peptide consisting of amino acids from the integrin binding region. The modification sites were identified via LC/MS. Our research indicated that blue light irradiation caused cleavage throughout the A2E molecule closest to the pyridinium ring, and attached to the fibronectin peptide preferentially at lysine and arginine residues. All of these reactions are similar to the Maillard reaction. Altogether this study suggests that blue-light-irradiated A2E modifies peptides and forms advance glycation endproducts. Furthermore, these results can be used to identify modifications that occur in Bruch's membrane in vivo.