Expression Of Basement Membrane Proteins Research Articles

A biosynthetic alternative to human amniotic membrane for cornea repair—A comparative in‐vitro study

Aims/Purpose: We hypothesize that the biosynthetic Symatix membrane (SM) can replace human amniotic membrane (HAM) in ocular surgical applications. Our aim was to test this hypothesis by comparing the performance of limbal epithelial cells (LECs) cultures on the membranes.Methods: SM was manufactured by the Electrospinning Company Ltd using its proprietary Symatix® Technology platform. SM consists of degradable medical grade poly (lactic‐co‐glycolic acid) polymer fibres and sodium hyaluronan. The suturing and fibrin gluing of SM were tested ex‐vivo by simulation on human corneas. LECs from human limbal tissue were used for testing cellular properties namely cell proliferation (scratch wound and Ki‐67 assay), metabolic activity, limbal epithelial cell marker (cytokeratin‐19 (CK‐19)) tight junction formation (zonula occludens‐1 (ZO‐1)), cell‐ECM interaction (fibronectin (FN)), and basement membrane protein expression (laminin) on SM, plastic, HAM, and freeze‐dried amniotic membrane (FDAM).Results: The surgical handleability of SM was equivalent to HAM. Histological studies demonstrated that epithelial cells on SM had the typical tightly apposed, polygonal, corneal epithelial cell morphology. Rapid wound healing occurred on SM within 3 days; Ki‐67 revealed increased progressive proliferation; and improved metabolic activity was found on SM compared to HAM and FDAM. Immunofluorescence studies showed positive expression of CK‐19, collagen‐1 (Col‐1), laminin, ZO‐1, and FN in SM, plastic, and FDAM compared to negative expression of CK‐19 and ZO‐1 and positive expression of collagen‐1 (Col‐1), laminin and FN in HAM. This could indicate that SM is a better substrate for epithelial cell migration, proliferation, and tight junction formation.Conclusions: This data demonstrates that SM can provide a suitable alternative to HAM for surgical application in sight‐restoring operations.

The matrix microenvironment influences but does not dominate tissue-specific stem cell lineage differentiation

Mesenchymal stem cells (MSCs) play a pivotal role in tissue engineering and regenerative medicine, with their clinical application often hindered by cell senescence during ex vivo expansion. Recent studies suggest that MSC-deposited decellularized extracellular matrix (dECM) offers a conducive microenvironment that fosters cell proliferation and accentuates stem cell differentiation. However, the ability of this matrix environment to govern lineage differentiation of tissue-specific stem cells remains ambiguous. This research employs human adipose-derived MSCs (ADSCs) and synovium-derived MSCs (SDSCs) as models for adipogenesis and chondrogenesis differentiation pathways, respectively. Genetically modified dECM (GMdECM), produced by SV40LT-transduced immortalized cells, was studied for its influence on cell differentiation. Both types of immortalized cells displayed a reduction in chondrogenic ability but an enhancement in adipogenic potential. ADSCs grown on ADSC-deposited dECM showed stable chondrogenic potential but increased adipogenic capacity; conversely, SDSCs expanded on SDSC-generated dECM displayed elevated chondrogenic capacity and diminished adipogenic potential. This cell-dependent response was confirmed through GMdECM expansion, with SDSCs showing enhanced chondrogenesis. However, ADSCs did not exhibit improved chondrogenic potential on GMdECM, suggesting that the matrix microenvironment does not dictate the final differentiation path of tissue-specific stem cells. Potential molecular mechanisms, such as elevated basement membrane protein expression in GMdECMs and dynamic TWIST1 expression during expansion and chondrogenic induction, may underpin the strong chondrogenic differentiation of GMdECM-expanded SDSCs.

The role of transforming growth factor- in hypertension-induced cerebrovascular remodeling

Background: Hypertension (HT) promotes structural and functional changes in the cerebral microcirculation that can provoke irreversible cerebrovascular injury, leading to neuronal loss and brain atrophy,1 cognitive impairment, vascular dementia,2 and Alzheimer’s disease.3 Currently, the mechanisms and consequences of such remodeling are not fully understood. Transforming growth factor (TGF) is a morphogen that regulates cellular differentiation, induces endothelial-to-mesenchymal transition (EndoMT), and works as a contractile-to-synthetic switch in vascular smooth muscle cells (VSMC) phenotype.4 Plasma levels of TGF are increased in HT patients, and in spontaneously hypertensive rats (SHR) that exhibit vascular fibrosis.5 Although coincidental, causal roles through which elevated TGF may drive cerebrovascular remodeling in the HT brain are suspected, but unproven. We hypothesize that HT-induced TGF drives cerebrovascular remodeling, which decreases blood-brain barrier and impairs cerebrovascular autoregulation.
 Methods: Brain cortices and penetrating cerebral microvessels (BMVs) were isolated from male and female SHR and Wistar-Kyoto rats (WKY, control), and subjected to western blotting and immunofluorescence labeling for endothelial cell (EC) and VSMC differentiation markers, TGF canonical pathway markers SMAD2, 3 and 4, as well as basement membrane protein expression, and glial fibrillary acidic protein (GFAP) as a marker of astrocyte inflammation. Additionally, TGF-treated rat retinal microvascular endothelial cells (RRMECs), and human brain microvascular endothelial cells (hCMEC/D3) ± the TGF inhibitor vactosertib, were also subjected to western blotting and immunofluorescence labeling to assess endothelial cell (EC) and VSMC differentiation markers, as well as basement membrane protein expression.
 Results: TGF was significantly increased in SHR BMVs. Moreover, GFAP levels were significantly increased in the SHR cortex. The EC junctional proteins platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin), were significantly decreased in SHR. In contrast, CRBP-1, a marker for synthetic VSMC, and collagen IV and fibronectin levels significantly increased in SHR. Interestingly, female SHR rats had significantly increased levels of SMAD2/3, which was not evident in male SHRs, indicating a possible role for the non-canonical TGF pathway in male SHRs. Additionally, TGF significantly increased -smooth muscle actin (-SMA) and decreased VE-cadherin expression in RRMECs and D3 cells, consistent with EndoMT. TGF inhibition with vactosertib reversed these effects and significantly suppressed -SMA, and maintained VE-Cadherin similar to control RRMECs and D3s.
 Conclusions: We now have strong evidence for HT-induced cerebrovascular remodeling, where TGF mediates loss of both smooth muscle and endothelial differentiated phenotypes. The reversal of these effects using TGF blockers suggests that therapy to modulate TGF may represent a means to control HT-induced cerebrovascular remodeling.

Gestational folic acid deficiency alters embryonic eye development: Possible role of basement membrane proteins in eye malformations

ObjectivesFolic acid (FA) is crucial before and during early pregnancy. FA deficiency can occur because dietary FA intake is low in mothers at the time of conception. Likewise, various ocular pathologies are related to the alteration of extracellular matrices. The present study aimed to investigate the association between maternal FA deficiency and congenital eye defects. We also investigated whether maternal diet deficient in FA alters the expression of collagen IV and laminin-1 as a possible mechanism responsible for the appearance of ocular malformations. Both proteins are the main components of the basal lamina, and form an interlaced network that creates a relevant scaffold basement membrane. Basal laminae are involved in tissues maintenance and implicated in regulating many cellular processes. MethodsA total of 57 mouse embryos were classified into the following groups: Control group, (mothers were fed a standard rodent diet), and D2 and D8 groups (mothers were fed FA-deficient [FAD] diet for 2 or 8 wk, respectively). Female mice from group D2 were fed a FAD diet (0 mg/kg diet + 1% succinyl sulfathiazole used to block the synthesis of FA) for 2 wk from the day after mating until day 14.5 of gestation (E14.5). On the other hand, female mice from group D8 were fed a FAD diet for 8 wk (6 wk before conception and during the first 2 wk of pregnancy). For the data analysis, we first estimated the incidence of malformations in each group. Then, the statistical analysis was performed using IBM SPSS Statistics, version 25.0. Expression patterns of collagen IV and laminin-1 were examined with the immunohistochemical technique. ResultsOur results showed that mice born to FA-deficient mothers had several congenital eye abnormalities. Embryos from dams fed a short-term FAD diet were found to have many significant abnormalities in both anterior and posterior segments, as well as choroidal vessel abnormalities. However, embryos from dams fed a long-term FAD diet had a significantly higher incidence of eye defects. Finally, maternal FA deficiency increased the expression of both collagen IV and laminin-1. Likewise, changes in the spatial localization and organization of collagen IV were observed. ConclusionsA maternal FAD diet for a short-term period causes eye developmental defects and induces overexpression of both collagen IV and laminin-1. The malformations observed are probably related to alterations in the expression of basement membrane proteins.

Intravenous Injection of Muse Cells as a Potential Therapeutic Approach for Epidermolysis Bullosa

Intravenous Injection of Muse Cells as a Potential Therapeutic Approach for Epidermolysis Bullosa

Slit/Robo signals prevent spinal motor neuron emigration by organizing the spinal cord basement membrane.

The developing spinal cord builds a boundary between the CNS and the periphery, in the form of a basement membrane. The spinal cord basement membrane is a barrier that retains CNS neuron cell bodies, while being selectively permeable to specific axon types. Spinal motor neuron cell bodies are located in the ventral neural tube next to the floor plate and project their axons out through the basement membrane to peripheral targets. However, little is known about how spinal motor neuron cell bodies are retained inside the ventral neural tube, while their axons can exit. In previous work, we found that disruption of Slit/Robo signals caused motor neuron emigration outside the spinal cord. In the current study, we investigate how Slit/Robo signals are necessary to keep spinal motor neurons within the neural tube. Our findings show that when Slit/Robo signals were removed from motor neurons, they migrated outside the spinal cord. Furthermore, this emigration was associated with abnormal basement membrane protein expression in the ventral spinal cord. Using Robo2 and Slit2 conditional mutants, we found that motor neuron-derived Slit/Robo signals were required to set up a normal basement membrane in the spinal cord. Together, our results suggest that motor neurons produce Slit signals that are required for the basement membrane assembly to retain motor neuron cell bodies within the spinal cord.

Type IV Collagenase Inhibitor, BiPS, Alters Matrix Metalloproteinase 9 Expression in Sulfur Mustard Exposed Mouse Skin

Sulfur mustard (bis‐2‐chloroethylsulfide, SM), is a chemical warfare agent that penetrates the skin rapidly causing extensive blisters and constitutes a potentially serious threat to military and civilian populations. While its mechanisms of toxic action are yet poorly defined, animal models demonstrate disruption of the skin architecture at the dermal‐epidermal junction (DEJ). Collagen degradation in the basement membrane zone by matrix metalloproteinases (MMPs) further exacerbates vesication post SM exposure. The type IV collagenase (MMP9 and MMP2) may mediate the disruption of type IV collagen (Col IV), a main component of the basement membrane zone (BMZ) in skin wounds post SM exposure. N‐hydroxy‐3‐phenyl‐2‐(4‐phenylbenzenesulfonamido) propanamide (BiPS) was tested as a highly selective MMP2/MMP9 inhibitor. The study aimed to evaluate the therapeutic effectiveness of topically delivered BiPS to protect against SM induced skin injury. We performed a time course study (24, 72, and 168 hrs post‐SM exposure) and a compound evaluation study (BiPS as a single pretreatment using the same time points post SM exposure) on mouse ear skin to determine improvement using histopathologic changes, MMP9 gene expression, and basement membrane protein expression as markers. SM induced characteristic skin structural damage including edema, inflammatory cell infiltration, and epidermal hyperplasia. MMP9 mRNA expression increased with time. Dual immunofluorescent studies using Col IV α1 and MMP9 antibodies showed BMZ breakdown of Col IV in the basement membrane and apparent MMP9 expression in the epidermis and the adjacent dermal matrix of the SM injured skin. In the compound evaluation study, the BiPS pretreatment group demonstrated significant reduction in edema and improvement in overall tissue structure by 72 hr post‐SM exposure. mRNA expression of MMP9 was significantly downregulated in the BiPS treated group by 168 hr post exposure. Immunofluorescent studies showed that significant reduction of MMP9 expression in the skin sections for all time points; the basement membrane appeared improved as indicated by a continuous (without disruption) expression of Col IV in the BMZ; which was similar to the unexposed control skin. These results suggest that BiPS may modulate MMP9 expression which may in turn improve basement membrane integrity after skin exposure to SM. BiPS, the MMP2/MMP9 inhibitor II, may present as a potential therapeutic countermeasure against SM induced skin injury.Support or Funding InformationES005022, T32ES007148, and NIAMS U54AR055073This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aberrant Patterns of Key Epithelial Basement Membrane Components in Keratoconus.

In the cornea, the epithelial basement membrane (BM) plays an important role in maintaining corneal integrity and homeostasis. Aberrations in this vital structure are associated with several corneal pathologies including keratoconus. The aim of this study was to investigate the expression of key structural components of the epithelial BM in keratoconic corneas and to identify and describe any aberrant patterns. Immunohistochemical labeling of key BM components including fibronectin, laminin, and type IV and VII collagen was performed in healthy and keratoconic corneas. Clear changes in the BM components in the keratoconic corneas were seen with the key structural components either being absent or forming a discontinuous pattern. Another aberrant pattern, the expression of BM proteins, particularly fibronectin, laminin, and type IV collagen, in the anterior stroma of keratoconic corneas was also observed. These results indicate the activation of keratocytes into the fibroblast and myofibroblast wound phenotypes and the potential source of corneal scarring commonly observed in keratoconic corneas. Our data also support the hypothesis of dysregulated collagen synthesis and breakdown in the keratoconic cornea, in particular, the BM, and suggest a role for the BM in initiation and progression of keratoconus.

Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier.

The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different invitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)]2 α2(IV), agrin, perlecan, and nidogen 1 and 2 invitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured invitro coherently form a BBB and express basement membrane proteins as a feature of maturation. Cover Image for this issue: doi: 10.1111/jnc.13789.

Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts.

Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and CXCL8) was observed followed by complete reepithelialization. Seven days after wounding, tissue integrity, metabolic activity, and cytokine levels had returned to the prewounded state. In conclusion, immortalized human gingiva KC and fibroblasts can be used to make physiologically relevant GE, which resemble either the healthy gingiva or a neoplastic disease model. These organotypic models will provide valuable tools to investigate oral mucosa biology and can also be used as an animal alternative for drug targeting, vaccination studies, microbial biofilm studies, and testing new therapeutics.

Transcriptome Analysis of HepG2 Cells Expressing ORF3 from Swine Hepatitis E Virus to Determine the Effects of ORF3 on Host Cells.

Hepatitis E virus- (HEV-) mediated hepatitis has become a global public health problem. An important regulatory protein of HEV, ORF3, influences multiple signal pathways in host cells. In this study, to investigate the function of ORF3 from the swine form of HEV (SHEV), high-throughput RNA-Seq-based screening was performed to identify the differentially expressed genes in ORF3-expressing HepG2 cells. The results were validated with quantitative real-time PCR and gene ontology was employed to assign differentially expressed genes to functional categories. The results indicated that, in the established ORF3-expressing HepG2 cells, the mRNA levels of CLDN6, YLPM1, APOC3, NLRP1, SCARA3, FGA, FGG, FGB, and FREM1 were upregulated, whereas the mRNA levels of SLC2A3, DKK1, BPIFB2, and PTGR1 were downregulated. The deregulated expression of CLDN6 and FREM1 might contribute to changes in integral membrane protein and basement membrane protein expression, expression changes for NLRP1 might affect the apoptosis of HepG2 cells, and the altered expression of APOC3, SCARA3, and DKK1 may affect lipid metabolism in HepG2 cells. In conclusion, ORF3 plays a functional role in virus-cell interactions by affecting the expression of integral membrane protein and basement membrane proteins and by altering the process of apoptosis and lipid metabolism in host cells. These findings provide important insight into the pathogenic mechanism of HEV.

Promise of Human Induced Pluripotent Stem Cells in Skin Regeneration and Investigation

Induced pluripotent stem cell (iPSC) technology has initiated a new era in biomedical science. The skin has been realized as an ideal platform for iPSC applications; unlike other organs, the skin is easily accessible, highly proliferative, and reconstitutable. Currently, skin equivalents can be generated from iPSCs not only from healthy individuals but also from patients with genodermatoses, providing novel platforms for dissecting disease pathophysiology and establishing cell-based therapy. With their developmental plasticity, iPSCs may also enable the regeneration of skin appendages. The iPSC technology may provide novel remedies for intractable disorders, once key issues particularly, safety concerns, are cleared.

Curcumin ameliorates intrahepatic angiogenesis and capillarization of the sinusoids in carbon tetrachloride-induced rat liver fibrosis

Neoangiogenesis and the development of an abnormal angio-architecture in the liver are strongly linked with progressive fibrogenesis. This study aimed to evaluate the ability of curcumin to protect liver fibrosis-associated angiogenesis and capillarization of the sinusoids in experimental rats. Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4) with or without curcumin for 6 weeks. The results suggest that curcumin treatment markedly attenuated CCl4-induced liver fibrosis, as assessed by histology and hydroxyproline content, and inhibited hepatic stellate cell activation. Curcumin ameliorated hepatic angiogenesis, as assessed by measuring microvessel density using Von Willebrand factor staining and by examining the expression of the endothelial cell markers CD31 and vascular endothelial growth factor receptor (VEGFR)-2 in the livers. Pathologic remodeling of liver sinusoidal capillarization, as assessed by electron-microscopic analysis of Disse's space and by evaluation of the levels of basement membrane protein expression, was also attenuated by curcumin administration. The intrahepatic gene or protein expression of hypoxia-inducible factor-1α, VEGFR-1, placental growth factor, and cyclooxygenase-2 decreased with treatment with curcumin in fibrotic rats. In conclusion, curcumin ameliorates hepatic angiogenesis and sinusoidal capillarization in CCl4-induced rat liver fibrosis through suppressing multiple proangiogenic factors.

Blood basement membrane alterations in human retinal microaneurysms during aging

AbstractPurpose Different studies indicates that the presence of retinal microaneurysms, dilations of the capillaries which often develop as gross outpouchings of the vessel wall, appears during human aging. However, little is known about the mechanisms involved in the development of these structures. The aim of this study was to examine whether basement membrane protein expression and organization changes during the formation of retinal microaneuryms.Methods Human retinas were obtained from 17 donors: 14 old‐donors and 3 middle‐aged donors. Basement membrane proteins (collagen IV, laminin, fibronectin, nidogen), perlecan and MMP‐9 expression were analyzed in paraffin sections and whole‐mount formalin fixed retinas using immunohistochemistry and laser‐confocal microscopy. The structure of basement membrane was analyzed by TEM.Results Microaneurysms were present in all the retinas obtained from old donors but were absent in the retinas obtained from midle‐aged donors. Microaneurysms were classified as Type I (early stage) and Type II (advanced stage). We observed that all basement membrane proteins increased its expression in Type I microaneurysms, but they disminished in Type II microaneuryms. In addition, MMP‐9 was strongly expressed in microaneurysms producing basement membrane disorganization.Conclusion Collagen IV, laminin, fibronectin, nidogen and perlecan were increased in Type I microaneurysm basement membrane. A concommitant expression of MMP‐9 could explain the disorganization of blood basement membrane components.

Disruption of Arterial Perivascular Drainage of Amyloid-β from the Brains of Mice Expressing the Human APOE ε4 Allele

Failure of elimination of amyloid-β (Aβ) from the brain and vasculature appears to be a key factor in the etiology of sporadic Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). In addition to age, possession of an apolipoprotein E (APOE) ε4 allele is a strong risk factor for the development of sporadic AD. The present study tested the hypothesis that possession of the APOE ε4 allele is associated with disruption of perivascular drainage of Aβ from the brain and with changes in cerebrovascular basement membrane protein levels. Targeted replacement (TR) mice expressing the human APOE3 (TRE3) or APOE4 (TRE4) genes and wildtype mice received intracerebral injections of human Aβ40. Aβ40 aggregated in peri-arterial drainage pathways in TRE4 mice, but not in TRE3 or wildtype mice. The number of Aβ deposits was significantly higher in the hippocampi of TRE4 mice than in the TRE3 mice, at both 3- and 16-months of age, suggesting that clearance of Aβ was disrupted in the brains of TRE4 mice. Immunocytochemical and Western blot analysis of vascular basement membrane proteins demonstrated significantly raised levels of collagen IV in 3-month-old TRE4 mice compared with TRE3 and wild type mice. In 16-month-old mice, collagen IV and laminin levels were unchanged between wild type and TRE3 mice, but were lower in TRE4 mice. The results of this study suggest that APOE4 may increase the risk for AD through disruption and impedance of perivascular drainage of soluble Aβ from the brain. This effect may be mediated, in part, by changes in age-related expression of basement membrane proteins in the cerebral vasculature.

Altered Collagen Production by Dysfunctional Mesenchymal Stem Cells Is Linked to T Cell Large Granular Lymphocyte Leukemia Pathophysiology.

AbstractAbstract 2595Large Granular Lymphocyte (LGL) leukemia is a chronic lymphoproliferative syndrome that can be broadly classified into two groups depending on whether the expanded cells are T-cells or NK-cells. The clinical characteristics of the disease include lymphocytosis, neutropenia, anemia, that can be associated with rheumatoid arthritis and pulmonary arterial hypertension (PAH). Hematologic improvement with immunosuppressive agents such as cyclosporine and low-dose methotrexate has lead to the widely accepted theory that cytopenias are mediated by autoimmune destruction of the hematopoietic stem cell (HSC) compartment or lysis of mature myeloid cells in circulation. We found, however, that autologous HSCs and mature granulocyte populations fail to be recognized or lysed ex vivo by T-LGL leukemia cells suggesting that an alternate mechanism may be involved. In contrast to research done on the T-LGL cells themselves, the role of the bone marrow microenvironment and HSC compartment in T-LGL leukemia patients is completely unexplored. Therefore, bone marrow core biopsies, aspirates, and peripheral blood smears were obtained from 22 patients with LGL leukemia and 14 patients with non-hematological malignancies to serve as controls. Morphology and extracellular matrix composition were examined by H&E and reticulin stains, respectively. Utilizing the European consensus guidelines, grading scale (0 – 3), we determined that bone marrow reticulin fibrosis is present in patients with T-LGL leukemia (Figure 1). The mean fibrosis grade for the LGL group was 2.32 (median of 2.5), whereas, the mean fibrosis grade for the control group was 1.46 (median of 1.50), p-value of 0.01. Our analysis revealed that reticulin fibrosis in LGL leukemia bone marrow was particularly associated with the interstitial stroma and lymphoid aggregates. The degree of fibrosis in T-LGL bone marrow showed no relation to absolute neutrophil counts. However, an in depth analysis of neutrophil morphology revealed several dysplastic features within the neutrophil compartment in T-LGL patients. These features include decreased segmentation, increased numbers of pseudo-Pelger-Huet forms, and an increase percentage of immature neutrophils. Of these, the proportion of immature neutrophils positively correlated with fibrosis grade in T-LGL patients (Spearman r=0.7302; p=0.0002), indicating a possible link between reticulin fibrosis and the quality of hematopoiesis. To explore the pathogenesis of medullary fibrosis, mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of 6 LGL leukemia patients and 5 healthy controls and then expanded ex vivo under non-differentiating conditions. During expansion, healthy MSCs produce cytokines and growth factors necessary for self renewal and for the support of hematopoiesis. However, MSCs from T-LGL patients displayed severely reduced self-renewal potential, reaching a mean of 7 population doublings compared to a mean of 23 for normal MSCs, and were unable to support the proliferation of healthy HSCs in a co-culture proliferation assay. Microarray analysis (H6 V133 plus 2.0) was performed on the MSCs from both control and T-LGL patients with analysis focused on genes regulating basement membrane composition. For normal MSCs, significant reductions in the expression of numerous collagen genes occured as the cells underwent expansion in self-renewal conditions. However, MSCs from T-LGL patients failed to downregulate these genes despite months of culture. The most prominent collagen genes following this pattern were types I (α1, α2), III (reticulin), IV (α1, α2), and V (α1, α2). A combination of qRT-PCR and immunflourescent staining (Figure 2) were utilized to confirm these gene expression changes. Collectively, these results implicate aberrant MSC self-renewal capacity and skewed basement membrane protein expression in the pathogenesis of T-LGL leukemia and suggest that these abnormalities may represent novel targets for future drug discovery. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture

We describe, for the first time, the use of cellular plastic compressed collagen as a substrate for human limbal epithelial cell expansion and stratification. The characteristics of expanded limbal epithelial cells on either acellular collagen constructs or those containing human limbal fibroblasts were compared to a human central cornea control. After compression, human fibroblasts in collagen constructs remained viable and limbal epithelial cells were successfully expanded on the surface. After airlifting, a multilayered epithelium formed with epithelial cell morphology very similar to that of cells in the central cornea. Immunochemical staining revealed expression of basement membrane proteins and differentiated epithelial cell markers found in native central cornea. Ultrastructural analysis revealed cells on collagen constructs had many features similar to central cornea, including polygonal, tightly opposed surface epithelial cells with microvilli and numerous desmosomes at cell–cell junctions. Taken together, these data demonstrate that plastic compressed collagen constructs can form the basis of a biomimetic tissue model for in vitro testing and could potentially provide a suitable alternative to amniotic membrane as a substrate for limbal epithelial cell transplantation.

Design and testing of biological scaffolds for delivering reparative cells to target sites in the lung

This study summarizes the development and testing of a scaffold to promote engraftment of cells in the distal lung. A fibrinogen-fibronectin-vitronectin hydrogel (FFVH) was developed and optimized with respect to its mechanical and biological properties for this application. In vitro, FFVH scaffolds promoted attachment, histiotypic growth and expression of basement membrane proteins by primary ovine lung mesenchymal cells derived from lung biopsies. In vivo testing was then performed to assess the ability of FFVHs to promote cell engraftment in the sheep lung. Treatment with autologous cells delivered using FFVH was clinically well tolerated. Cells labelled with a fluorescent dye (PKH-26) were detected at treatment sites after 1 month. Tissue mass (assessed by CT imaging) and lung perfusion (assessed by nuclear scintigraphy) were increased at emphysema test sites. Post-treatment histology demonstrated cell proliferation and increased elastin expression without scarring or collapse. No treatment-related pathology was observed at healthy control sites. FFVH scaffolds promote cell attachment, spreading and extracellular matrix expression in vitro and apparent engraftment in vivo, with evidence of trophic effects on the surrounding tissue. Scaffolds of this type may contribute to the development of cell-based therapies for patients with end-stage pulmonary diseases.

Promotion of Tumor Cell Migration by Extracellular Matrix Proteins in Human Pancreatic Cancer

The pathogenesis of pancreatic carcinoma is driven by the tumor cells ability to migrate causing invasion and metastases. The correlation between the aberrant expression of basement membrane proteins and the process of tumor invasion and metastasis has not been fully determined. In the present study, the influence of laminin, fibronectin, and collagen type IV on migratory activity of 5 different cell lines has been investigated at the level of a single tumor cell using 3-dimensional time-lapse microscopy. All investigated cell lines have shown a high baseline migration that varied between 6.2 +/- 3.6 and 20.6 +/- 6.8 microm/h. The addition of laminin, fibronectin, and collagen type IV to collagen type I matrix has significantly increased tumor cell migration. Tumor cell migration was strongly inhibited after treating the tumor cells with anti-beta1 monoclonal antibodies. An abundant and continuous expression of laminin, fibronectin, and collagen type IV was found on the basement membrane of perineurium, which sharply promoted tumor cell invasion. The continuous presentation of the basement membrane proteins by perineurium contributes to the affinity of pancreatic cancer cells for the perineural tumor invasion. Blockade of integrins could represent a possible approach to control the basement membrane-guided tumor spread.

Construction of Synthetic Dermis and Skin Based on a Self-Assembled Peptide Hydrogel Scaffold

Using biocompatible peptide hydrogel as a scaffold, we prepared three-dimensional synthetic skin that does not contain animal-derived materials or pathogens. The present study investigated preparation methods, proliferation, and functional expression of fibroblasts in the synthetic dermis and differentiation of keratinocytes in the epidermis. Synthetic dermis was prepared by mixing fibroblasts with peptide hydrogel, and synthetic skin was prepared by forming an epidermal layer using keratinocytes on the synthetic dermis. A fibroblast-rich foamy layer consisting of homogeneous peptide hydrogel subsequently formed in the synthetic dermis, with fibroblasts aggregating in clusters within the septum. The epidermis consisted of three to five keratinocyte layers. Immunohistochemical staining showed human type I collagen, indicating functional expression around fibroblasts in the synthetic dermis, keratinocyte differentiation in the epidermis, and expression of basement membrane proteins. The number of fibroblasts tended to increase until the second week and was maintained until the fourth week, but rapidly decreased in the fifth week. In the synthetic dermis medium, the human type I collagen concentration increased after the second week to the fifth week. These findings suggest that peptide hydrogel acts as a synthetic skin scaffold that offers a platform for the proliferation and functional expression of fibroblasts and keratinocytes.