Laminin Subunit Alpha Research Articles

Breaking the ice: urine proteomics of medullary sponge kidney disease

Urinary proteomics is a promising tool for biomarker investigation, particularly in complex kidney diseases. Fabris and colleagues report that urinary laminin subunit alpha-2 is a potential diagnostic marker of medullary sponge kidney (MSK) disease by using a label-free quantitative proteomics platform and a clinically compatible enzyme-linked immunosorbent assay. The neglected issue of stone pathogenesis was also evidenced. This commentary discusses several considerations in biomarker validation, and how urinary proteomics breaks new ground in MSK research.

P3.03-039 Prognostic Biomarkers for Malignant Pleural Mesothelioma Treated with Chemotherapy: Topic: Mesothelioma Clinical

Prognosis of Malignant Pleural Mesothelioma (MPM) is poor and median survival is about 12 months. If any associations can be established between biomarkers and MPM, there will be benefit to clinical practice. In this study, the aim is to examine expression levels of the genes selected from relevant literature and utilizing in silico methods in the determination of prognosis of MPM. The study group consisted of 54 MPM patients treated by chemotherapy. The expression of 6 genes; sestrin 1 (SESN1), laminin subunit alpha 4 (LAMA4), midkine (MKN), fibulin-3 (EFFL-3), syndecan-1 (SOC-1) and hyaluronan-2 (HYA-2) were examined by qPCR in the tumor tissues. SESN1 and LAMA4 were identified using an in house R based script “Unsupervised Survival Analysis Tool.” MKN, EFFL-3, SOC1 and HYA-2 were determined according to existing literature. We used two housekeeping genes; glucose-6-phosphate dehydrogenase (G6PD), TATA-box binding protein (TBP) as controls. qPCR Relative quantification of gene expression was based on the geometric mean G6PD, TBP. The relation between gene expression and prognosis was determined by categorizing samples into two groups using all possible cut-off values, analyzed by the Log Rank test (Log-rank test with multiple cut-offs). Cut-offs generating a p value less than 0.05, between %10-90 percentiles were considered significant. Mean age of study group was 62.5± 9.7 years (r:36-82). Of the patients, 43 (79.6%) had epithelioid cell type mesothelioma. The median survival (MS) for all patients was 10 (±1.18 SE) months (CI 95%;7.69 to 12.30). Twenty-five patients (46.3%) survived less than 12 months, 29 (53.7%) more than 12 months, and 4 (7.4%) were still surviving at the end of the study. The clinical factors that was associated with survival were histopathology (p=0.004) and stage (p=0.036). MKN, EFFL-3, SOC1, HYA-2, SESN-1 were found to be associated with survival time by univariate analyses. After the correction with histopathology and stage, MKN (p=0.041), SOC1 (p=0.015), HYA-2 (p=0.003), SESN-1 (p=0.028) were found to be related with survival time. Additionally, in only epithelioid type MPM patients, HYA-2 expression was found to be related with survival time according to multivariate analyses (p=0.016). High MKN expression is potential biomarker of poor prognosis and high SOC1, HYA-2, SESN-1 expressions are potential biomarkers of good prognosis in MPM patients, and should be further investigated. High HYA-2 expression also can be utilized as good prognosis biomarker for epithelioid type MPM. *This study was partly supported by General Directorate of Health Researches, Republic of Turkey, Ministry of Health.

Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160.

Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis. We thus developed an alternative method to laminin-111 protein repeated administration by inducing expression of the endogenous mouse Lama1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the Lama1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. Lama1 mRNA (qRT-PCR) and proteins (immunohistochemistry and western blot) were not detected in the control C2C12 myoblasts and in control muscles. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 and a gRNA. Larger synergic increases were observed by using two or three gRNAs. The increased Lama1 expression did not modify the expression of the α7 and β1 integrins. Increased expression of Lama1 by the CRISPR/Cas9 system will have to be further investigated by systemic delivery of the CRISPR/Cas9 components to verify whether this could be a treatment for several myopathies.

RNA Sequencing of Bone Marrow Derived Mesenchymal Stromal Cells (MSCs) of Healthy Young, Healthy Old and Patients with Myelodysplastic Syndrome (MDS) Reveals Transcriptomic Changes upon Aging of the Niche and Onset of MDS

Introduction: Myelodysplastic Syndrome (MDS) can occur in young people but it is mainly a disease of the elderly with a dramatic increase of incidence in the decades above 60 years. Accordingly, the factor age may be an important gateway to the understanding of the molecular pathogenesis of MDS. Insights into the molecular changes of aging hematopoiesis in healthy organisms have found molecular changes, which often parallel the observations in MDS such as increase of clonality with age, change of epigenetic profiles, skewed lineage commitment toward the myeloid compartment and reduced regenerative capacity after stress. The development of MDS is often suggestive of an accelerated extrapolation of molecular changes, which also occur in normal aging hematopoiesis. Beyond this, increasing evidence is suggesting that MDS hematopoiesis is highly dependent on support of the bone marrow (BM) stroma, which has been shown to display aberrant transcriptomic profiles as compared to healthy BM stroma. To this end, we aimed to test the hypothesis whether the emergence of MDS may be associated with a continuity of molecular changes in BM stroma cells during aging. Therefore, we performed explorative RNA sequencing in a set of MSCs collected from healthy young, healthy old and patients with MDS with a highly standardized pre-analytical work-up algorithm.Methods: We collected BM samples from voluntary healthy young adults (age = 24 - 25 years, female n=3, male n=3), healthy old adults (age 66 - 79 years, female n=3, male n=3) and patients with very low - intermediate risk MDS (age 51 - 87 years, female n=3, male n=3). After isolation of BM mononuclear cells by Ficoll gradient centrifugation, 5x106 mononuclear BM cells were seeded into 25cm² flasks and cultured using StemMACS human MSC Expansion Media (Miltenyi Biotec) with weekly media exchange to select for MSCs. These were expanded and harvested in passage 2. Absence of residual hematopoietic cells was controlled by FACS with anti CD45, CD31, and CD146. Whole transcriptome RNA-sequencing on all samples was carried out from 150ng of high quality RNA using the TruSeq stranded total RNA protocol and 100bp paired end sequencing (Illumina). The bio-informatical pipeline consisted of mapping using hisat2 and cufflinks for calculation of differentially expressed genes.Results: RNA-sequencing generated a mean of 94 million reads per sample. Between the groups "healthy young" and "healthy old" 331 differentially regulated genes were identified. Between "healthy old" and "MDS" 514 genes were differentially regulated (fold change > 1.5, false discovery rate, FDR < 0.05). Among these, 197 genes were differently expressed between all three groups. With these parameters, a total of 17 genes showed a continuous and significant increase of expression from healthy young over healthy old toward MDS. Among these were Kit ligand (KITLG) but also a cluster of membrane based cell adhesion molecules such as Cadherin-6 (CDH6), Laminin Subunit Alpha 2 (LAMA2) and Laminin Subunit Gamma 2 (LAMC2) and others. Conversely, 5 genes showed a continuous and significant decrease of expression from healthy young over healthy old toward MDS, among these Leukocyte-specific protein 1 (LSP1), a gene implicated in regulation of T-cell migration. Gene set enrichment analysis revealed that MDS MSCs exhibited a significant depletion of genes involved in early adipogenic differentiation and enrichment of gene sets associated with extracellular matrix remodeling (FDR < 0.05, normalized enrichment score > 1.7). Although cells were cultured under normoxic conditions, MDS-MSCs displayed marked intrinsic feature of hypoxia.Conclusion: By integrating transcriptomic data from BM stroma cells from healthy individuals during aging and comparison to BM stroma cells from MDS patients we have identified gene sets that are significantly differentially expressed per continuitatem. On the background of the hypothesis that molecular changes in the microenvironment of MDS are an exacerbation of changes also taking place during normal aging in the bone marrow, these genes, which are accumulated in the context of extracellular matrix and cell adhesion are promising candidates to further elucidate a BM stroma based pathogenesis of MDS. DisclosuresNo relevant conflicts of interest to declare.

Proteomic-based research strategy identified laminin subunit alpha 2 as a potential urinary-specific biomarker for the medullary sponge kidney disease

Medullary sponge kidney (MSK) disease, a rare kidney malformation featuring recurrent renal stones and nephrocalcinosis, continues to be diagnosed using expensive and time-consuming clinical/instrumental tests (mainly urography). Currently, no molecular diagnostic biomarkers are available. To identify such we employed a proteomic-based research strategy utilizing urine from 22patients with MSK and 22 patients affected by idiopathic calcium nephrolithiasis (ICN) as controls. Notably, two patients with ICN presented cysts. In the discovery phase, the urine of 11 MSK and 10 controls, were randomly selected, processed, and analyzed by mass spectrometry. Subsequently, several statistical algorithms were undertaken to select the most discriminative proteins between the two study groups. ELISA, performed on the entire patients' cohort, was used to validate the proteomic results. After aninitial statistical analysis, 249 and 396 proteins were identified exclusive for ICN and MSK, respectively. A Volcano plot and ROC analysis, performed to restrict the number of MSK-associated proteins, indicated that 328 and 44 proteins, respectively, were specific for MSK. Interestingly, 119 proteins were found to differentiate patients with cysts (allpatients with MSK and the two ICN with renal cysts) from ICN without cysts. Eventually, 16 proteins were found to be common to three statistical methods with laminin subunit alpha 2 (LAMA-2) reaching the higher rank by a Support Vector Machine, a binary classification/prediction scheme. ELISA for LAMA-2 validated proteomic results. Thus, usinghigh-throughput technology, our study identified a candidate MSK biomarker possibly employable in future forthe early diagnosis of this disease.

Primary Linneg/CD45neg/CD271pos/PDGFRαlow/Neg Stroma Stem Cells In Adult Human Bone Marrow Show a Distinct Molecular Signature and Potent Hematopoiesis-Supporting Function

Human bone marrow contains a rare population of mesenchymal stroma stem cells (BM-MSC) that are capable of generating all skeletal lineages such as osteoblasts, adipocytes, chondrocytes and fibroblastic reticular cells. In vivo, BM-MSC are essential constituents of the hematopoietic stem cell niche, thus playing an important role in supporting, maintaining and controlling hematopoiesis. Despite their central role in bone marrow physiology, little is known about the primary MSC. This is mainly due to the fact that a precise phenotypical definition of BM-MSC has been lacking so far. We were first to report that low/negative expression of PDGFRα on linneg/CD45neg/CD271pos cells allowed identify the candidate primary stromal stem cell population in adult human bone marrow, i.e linneg/CD45neg/CD271pos/PDGFRαlow/neg cells were highly enriched in CFU-F (1 in 4) and showed all typical BM-MSC properties in-vitro and in-vivo (Li et al. Blood, 2012, 120:3460).This key finding now facilitated to characterize the molecular signature and hematopoiesis-supporting function of primary BM-MSC. Illumina Human HT-12 expression v4 BeadChips containing 48,107 probes were utilized to perform gene expression profiling analysis comparing linneg/CD45neg/CD271pos/PDGFRαlow/neg and linneg/CD45neg/CD271pos/PDGFRαpos cells (five independent samples in each group). The expression of 365 genes, including 22 novel surface antigens, such as ITGB5, ANTXR2, CD63, CD230, APLNR and CD74 was significantly higher in the PDGFRαlow/neg subset, whereas 65 genes showed reduced expression. The expression of cell cycle inhibitor genes, such as cyclin-dependent kinase inhibitor 1 (CDKN1A), B cell translocation gene family, member 3 (BTG3) and dual specificity protein phosphatase 3 (DUSP3), was increased in the PDGFRαlow/neg population, indicating a quiescent status of the majority of the cells, which was confirmed by cell cycle analysis (Ki67/DAPI staining; 96.8 ± 2.9% in G0 phase, n=3). In accordance with the hematopoiesis maintenance function, expression of genes encoding ECM proteins, such as laminin subunit alpha-4 (LAMA4), adrenomedullin (ADM) and collagen type I alpha 1 (COL1A1) was also significantly higher in PDGFRαlow/neg cells. Additionally, quantitative RT-PCR analysis revealed that PDGFRαlow/neg cells expressed high levels of CXCL12, VCAM1 and osteopontin, i.e. genes known to be related to hematopoietic stem cell (HSC) supportive function.In order to test the hematopoiesis-supporting capacity of primary BM-MSC, 7-day co-culture experiments with human cord blood CD34+ cells were performed in SCF, TPO, and FL-supplemented serum-free medium. CD34+ cells were evenly distributed and mainly found in close contact with stromal cells when cocultured with linneg/CD45neg/CD271pos/PDGFRαlow/neg cells, compared to cultures with linneg/CD45neg/CD271neg/PDGFRαneg cells and linneg/CD45neg/CD271pos/PDGFRαpos cells. Co-culture with PDGFRαlow/neg cells did not only amplify total hematopoietic cells (38.8 ± 14.4 fold) but, importantly, also expanded CD34+ cells very effectively (10.2 ± 3.8 fold, compared to 2.4 ± 0.2, 3.6 ± 0.7, and 3.2 ± 0.8 fold for no stroma, CD271neg/PDGFRαneg, and PDGFRαpos cells, respectively; p<0.05, n=3). Moreover, the percentage of CD34+ cells was 2-3 fold higher in PDGFRαlow/neg co-cultures. Xenotransplantation experiments investigating the expansion of transplantable hematopoietic stem cells are currently under way.Taken together, primary linneg/CD45neg/CD271pos/PDGFRαlow/neg stroma stem cells show a distinct molecular signature and potent hematopoiesis-supporting function. The study of primary BM-MSC will lead to a better understanding of the nature and the physiological role of these cells in the human bone marrow in-situ. Disclosures:No relevant conflicts of interest to declare.

Lobaplatin arrests cell cycle progression, induces apoptosis and alters the proteome in human cervical cancer cell Line CaSki

Cervical cancer is one of the most common gynecologic tumors. There is an upward trend in the incidence. The objective of this research was to explore the effect of lobaplatin on cervical cancer CaSki cells proliferation, cell cycle and apoptosis and analysis of the differential expressed proteins of CaSki cells after exposed to lobaplatin. Our findings have shown that lobaplatin inhibits cell proliferations in human cervical cancer CaSki cells in dose- and time-dependent manner. Flow cytometry assay confirmed that lobaplatin affected cervical cancer cell survival by blocking cell cycle progression in S phase and G0/G1 phase and inducing apoptosis in dose- and time-dependent manner. Lobaplatin treatment reduced polypyrimidine tract-binding protein 2, ribose-phosphate pyrophosphokinase, hypothetical protein, terminal uridylyltransferase 7, ubiquitin specific protease 16 and heterogeneous nuclear ribonucleoprotein A2/B1 expression and increase zinc finger protein 91, zinc finger protein, C-X-C motif chemokine 10 precursor, stromal cell protein and laminin subunit alpha-4 expression. Some of the differentially expressed proteins may be associated with antitumor effect of lobaplatin. Lobaplatin showed a good antitumour activity in in vitro models of human cervical cancer cells. These results indicate that lobaplatin could be an effective chemotherapeutic agent in human cervical cancer treatment by inducing apoptosis, cell cycle arrest and changing many kinds of protein molecule expression level.

Studies of Patients with Anti‐Epiligrin Cicatricial Pemphigoid

We have recently identified patients with a form of cicatricial pemphigoid who have IgG anti-basement membrane autoantibodies directed against epiligrin, a laminin isoform closely related if not identical to laminin 5. These patients' autoantibodies bind the lower lamina lucida of human epidermal basement membrane and immunoprecipitate this laminin isoform from extracts and media of biosynthetically radiolabeled human keratinocytes. Immunoblot studies show that these patients' autoantibodies specifically bind the alpha subunit of this laminin (i.e., laminin subunit alpha 3). We have found no evidence of these autoantibodies in normal volunteers or patients with other bullous skin diseases (including those with other forms of CP). These studies have identified a group of patients with an acquired, autoimmune, subepidermal bullous disorder who have disease-specific autoantibodies directed against the alpha subunit of epiligrin/laminin 5. These findings correlate with prior reports showing that a monoclonal antibody directed against this laminin subunit induces detachment of keratinocytes from extracellular matrix in vitro as well as epidermis from human skin in situ. Together, these findings suggest that this laminin mediates attachment of basal keratinocytes to epidermal basement membrane and that autoantibodies directed against it may be pathogenic. Moreover, recent studies showing that subunits of this laminin isoform are mutated in some patients with Herlitz's junctional epidermolysis bullosa indicate that acquired or inherited abnormalities in this adhesion ligand are associated with skin diseases characterized by separation of epidermis from epidermal BM.

Anti-Basement Membrane Autoantibodies in Patients with Anti-Epiligrin Cicatricial Pemphigoid Bind the α Subunit of Laminin 5

Recent studies have identified a group of cicatricial pemphigoid patients who have IgG anti-basement membrane autoantibodies that recognize epiligrin, a set of disulfide-linked polypeptides closely related if not identical to laminin 5 (formerly called kalinin, nicein, or BM600). To further understand the pathophysiology of blister formation in these patients, we have sought to identify the specific polypeptide(s) targeted by their autoantibodies. Comparative studies show that sera from these patients (nine of nine), P1E1 monoclonal anti-epiligrin antibody, and polyclonal as well as monoclonal anti-laminin 5 antibodies immunoprecipitate the same set of disulfide-linked polypeptides from media of biosynthetically radiolabeled human keratinocytes. Moreover, sera from eight of nine patients with anti-epiligrin cicatricial pemphigoid immunoblot the alpha subunit of laminin 5 but show no reactivity to its beta or gamma subunits. In addition, circulating IgG from a representative patient was affinity-purified against the alpha subunit of laminin 5 and shown to bind the dermal side of 1 M NaC1 split skin in the same manner as autoantibodies from all patients with anti-epiligrin cicatricial pemphigoid. Sera from patients with bullous pemphigoid (n = 5), other forms of cicatricial pemphigoid (n = 5), epidermolysis bullosa acquisita (n = 4), or bullous systemic lupus erythematosus (n = 1) show no reactivity against any subunit of this laminin isoform in immunoprecipitation or immunoblot experiments. These findings correlate with prior reports showing that a monoclonal antibody directed against the alpha subunit of laminin 5 (i.e., laminin subunit alpha 3) induces detachment of human keratinocytes from extracellular matrix in vitro as well as epidermis from human skin in situ. Together, these studies suggest that laminin subunit alpha 3 mediates attachment of basal keratinocytes to epidermal basement membrane and that autoantibodies directed against it may be pathogenic.