Laminin Chains Research Articles

YIGSR, A Laminin-Derived Peptide, Dictates a Concentration-Dependent Impact on Macrophage Phenotype Response.

Macrophage immune cells play crucial roles in the inflammatory (M1) and regenerative (M2) processes. The extracellular matrix (ECM) composition, including presentation of embedded ligands, governs macrophage function. Laminin concentration is abundant in the basement membrane and is dependent on pathological state: reduced in inflammation and increased during regeneration. Distinct laminin ligands, such as IKVAV and YIGSR, have disparate roles in dictating cell function. For example, IKVAV, derived from the alpha chain of laminin, promotes angiogenesis and metastasis of cancer cells whereas YIGSR, beta chain derived, impedes angiogenesis and tumor progression. Previous work has demonstrated IKVAV's inflammation inhibiting properties in macrophages. Given the divergent role of IKVAV and YIGSR in interacting with cells through varied integrin receptors, we ask: what role does laminin derived peptide YIGSR play in governing macrophage function? We quantified the influence of YIGSR on macrophage phenotype in 2D and 3D via immunostaining assessments for M1 marker inducible nitric oxide synthase (iNOS) and M2 marker Arginase-1 (Arg-1). We also analysed the secretome of human and murine macrophage response to YIGSR via a Luminex bead assay. YIGSR impact on macrophage phenotype occurs in a concentration-dependent manner. At lower concentrations of YIGSR, macrophage inflammation was increased whereas, at higher concentrations of YIGSR the opposite effect was seen within the same time frame. Secretomic assessments also demonstrate that pro-inflammatory chemokines and cytokines were increased at low YIGSR concentrations in M0, M1, M2 macrophages while pro-inflammatory secretion was reduced at higher concentrations. YIGSR can be used as a tool to modulate macrophage inflammatory state within M1 and M2 phenotypes depending on the concentration of peptide. YIGSR's impact on macrophage function can be leveraged for the development of immunoengineering strategies in regenerative medicine and cancer therapy. The online version contains supplementary material available at 10.1007/s12195-024-00810-5.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell–matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

Abstract 575: Brain delivery of clinically suitable nanobioconjugates to inhibit glioblastoma growth through extracellular matrix-immune cell crosstalk

Abstract Introduction: Tumor growth, invasion, and escape from immune surveillance largely depend on cancer microenvironment. Laminins are trimeric proteins and essential components of glioblastoma (GBM) microenvironment/extracellular matrix (ECM). In brain glioma samples from 230 patients, we found a correlation between the overexpression of tumor ECM protein laminin-411 (α4β1γ1) and faster tumor recurrence with shorter patient survival. Laminin-411 is produced by endothelial cells, neutrophils, monocytes, platelets, lymphocytes, and glioma cells and can modulate the immune system. Novel nanotechnology approach to block trimeric laminin-411 and activate of brain local immune system with brain delivery of PD-1 checkpoint inhibitor was developed for future translational application. Methods: Nanobioconjugates (NBC) based on poly (β-L-malic acid, P), were synthesized, characterized and used to intravenously treat mice with intracranial syngeneic GL261 or CT-2A GBM. The lead NBCs P/PEG/LLL(40%)/AON(α4β1)(2.0%)/AP-2(2%) and P/PEG/LLL(40%)/AP-2(2%)/αPD-1(0.2%) contained antisense to laminin-411 α4 and β1 chains, or αPD-1 antibody as well as trileucine (LLL) peptide for endosomal escape and AP-2 peptide for BBB crossing and tumor cell targeting. CRISPR/Cas9 constructs were used to knockdown α4 and β1 laminin chains in GBM ex vivo. Flow cytometry and RNA-seq analyses were performed to evaluate treatment. Results: Laminin-411 depletion with CRISPR/Cas9 and multifunctional NBC in vivo treatment equally suppressed GBM growth and significantly prolonged animal survival. The brain privileged immune system was activated upon treatment with a significant increase of CD3+, CD8+ T cells, NK, IFNγ+ NK cells, and M1 macrophages. RNA-sec analyses after treatment with a combination of NBC suppressing laminin-411 and checkpoint PD-1 showed enhanced anti-tumor effect with upregulation of genes coding for apoptotic Caspase 3 and IFNγ, and reduction of proliferation markers EGFR, c-Myc and Ki-67. Conclusion: Study describes novel GBM treatment strategy via NBCs crossing blood-brain barrier and targeting critical ECM and immune components of tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma. Citation Format: Alexander V. Ljubimov, Rameshwar Patil, Hui Ding, Liron Israel, Eggehard Holler, Julia Y. Ljubimova, Tao Sun, Keith L. Black. Brain delivery of clinically suitable nanobioconjugates to inhibit glioblastoma growth through extracellular matrix-immune cell crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 575.

Laminin-111 mutant studies reveal a hierarchy within laminin-111 genes in their requirement for basal epithelial tissue folding

The process of morphogenesis carefully crafts cells into complex organ structures which allows them to perform their unique functions. During brain development, the neuroepithelial tissue must go through apical and basal folding which is mediated through the instruction of both intrinsic and extrinsic factors. While much is known about apical folding, the mechanisms that regulate basal folding are less understood. Using the highly conserved zebrafish midbrain-hindbrain boundary (MHB) as an epithelial tissue model, we have identified the basement membrane protein laminin-111 as a key extrinsic factor in basal tissue folding. Laminin-111 is a highly conserved, heterotrimeric protein that lines the basal surface of the neuroepithelium. Laminin-111 is comprised of one alpha, one beta, and one gamma chain encoded by the genes lama1, lamb1, and lamc1, respectively. Human mutations in individual laminin-111 genes result in disparate disease phenotypes; therefore, we hypothesized that each laminin gene would have a distinctive role in tissue morphogenesis. Using zebrafish mutants for laminin-111 genes, we found that each laminin chain has a unique impact on basal folding. We found that lamc1 is the most critical gene for MHB morphogenesis, followed by lama1, and finally lamb1a. This hierarchy was discovered via three-dimensional single cell shape analysis, localization of myosin regulatory light chain (MRLC), and with analysis of MHB tissue folding later in development. These findings are essential for development of novel techniques in tissue engineering and to elucidate differences in human diseases due to specific chain mutations.

Efficacy Of Intradermal Allogeneic Fibroblast Injections In Junctional Epidermolysis Bullosa

Objective — to assess the efficacy and safety of intradermal injections of allogeneic fibroblasts into non-healing wounds in a patient with junctional epidermolysis bullosa. Material and Methods — A 49-year-old patient with intermediate junctional epidermolysis bullosa was injected intradermally into the base of non-healing wounds with 1 mL suspension of allogeneic fibroblasts, which contained 5×106 cells/mL, 10×106 cells/mL, and 20×106 cells/mL. Immunofluorescence mapping exhibited reduced β3 chain of laminin 332 and collagen XVII expression in the basement membrane area. Paired erosions were injected with 2% albumin or saline solution. Results — At two weeks after treatment, wound areas reduced significantly, or 100% re-epithelialization occurred. Collagen XVII and β3 chain expression of laminin 332 increased at the dermal-epidermal junction. Conclusion — Our findings demonstrated that intradermal injections of allogeneic fibroblasts could be an effective therapeutic approach for treating small non-healing wounds in junctional epidermolysis bullosa.

Amniotic Membrane Scaffolds Support Organized Muscle Regeneration in A Murine Volumetric Muscle Defect Model.

Background:Current treatment for volumetric muscle loss is limited to muscle transfer or acellular collagen scaffold (ACS) therapies that are associated with donor site morbidity and nonfunctional fibrosis, respectively. The aim of this study is to assess the utility of amniotic membrane scaffold (AMS) for volumetric muscle loss treatment.Methods:Murine quadriceps defects were created and randomized to three groups (n = 5/group): untreated controls, ACS, and AMS. In vivo muscle regeneration volume was quantified by MRI and microcomputed tomography. Muscle explants were analyzed using standard histology and whole-mount immunofluorescence at 8 weeks.Results:The cross-sectional muscle regeneration ratio was 0.64 ± 0.3 for AMS, 0.48 ± 0.07 for ACS, and 0.4 0 ± 0.03 for controls as assessed by MRI (P = 0.09) and 0.61 ± 0.28 for AMS, 0.50 ± 0.06 for ACS, and 0.43 ± 0.04 for controls as assessed by microcomputed tomography (P = 0.2). Histologically, AMS demonstrated significantly higher cellular density (900 ± 2 70 nuclei/high powered field) than ACS (210 ± 36) and control (130 ± 4) groups (P = 0.05). Immunofluorescence for laminin (AMS 623 ± 11 versus ACS 339 ± 3 versus control 115 ± 7; P < 0.01) and myosin heavy chain (AMS 509 ± 7 versus ACS 288 ± 5 versus control 84 ± 5; P = 0.03) indicated greater organized muscle fiber formation with AMS.Conclusion:AMS mediated muscle healing was characterized by increased cellular infiltration and organized muscle formation when compared with controls and ACS.

Injection laryngoplasty of human adipose-derived stem cell spheroids with hyaluronic acid-based hydrogel improves the morphological and functional characteristics of geriatric larynx

AimAs the geriatric population increased, the need of treatment for laryngeal atrophy and dysfunction increased. This study was performed to evaluate the effects of injection of human adipose-derived stem cell (hASC) spheroid-loaded catechol-conjugated hyaluronic acid (HA-CA) hydrogel on therapeutic rejuvenation of the geriatric larynx.MethodsStem cell spheroids with hyaluronic acid-based hydrogel were injected into the laryngeal muscles of 18-month-old Sprague–Dawley rats. The effects of hASC spheroids were examined in the following four groups: SHAM, injected with PBS; GEL, injected with HA-CA hydrogel; MONO, injected with single hASCs in HA-CA hydrogel; and SP, injected with hASCs spheroids in HA-CA hydrogel. The rejuvenation efficacy in geriatric laryngeal muscle tissues at 12 weeks postinjection was evaluated and compared by histology, immunofluorescence staining, and functionality analysis.ResultsTotal myofiber cross-sectional area and myofiber number/density, evaluated by detection of myosin heavy chain with antibodies against laminin and fast myosin heavy chain, were significantly higher in the SP group than in the other groups. The lamina propria of the larynx was evaluated by alcian blue staining, which showed that the HA was increased significantly in the SP group compared to the other groups. In functional analysis, the glottal gap area was significantly reduced in the SP group compared to the other groups. The phase difference in the vocal fold during vibration was also smaller in the SP group than in the other groups, but the difference did not reach statistical significance.ConclusionInjection of hASC spheroids with hyaluronic acid-based hydrogel improves the morphological and functional characteristics of geriatric larynx.Graphical abstract

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Pericytes contribute to the islet basement membranes to promote beta-cell gene expression

β-Cells depend on the islet basement membrane (BM). While some islet BM components are produced by endothelial cells (ECs), the source of others remains unknown. Pancreatic pericytes directly support β-cells through mostly unidentified secreted factors. Thus, we hypothesized that pericytes regulate β-cells through the production of BM components. Here, we show that pericytes produce multiple components of the mouse pancreatic and islet interstitial and BM matrices. Several of the pericyte-produced ECM components were previously implicated in β-cell physiology, including collagen IV, laminins, proteoglycans, fibronectin, nidogen, and hyaluronan. Compared to ECs, pancreatic pericytes produce significantly higher levels of α2 and α4 laminin chains, which constitute the peri-islet and vascular BM. We further found that the pericytic laminin isoforms differentially regulate mouse β-cells. Whereas α2 laminins promoted islet cell clustering, they did not affect gene expression. In contrast, culturing on Laminin-421 induced the expression of β-cell genes, including Ins1, MafA, and Glut2, and significantly improved glucose-stimulated insulin secretion. Thus, alongside ECs, pericytes are a significant source of the islet BM, which is essential for proper β-cell function.

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.

Cellular and Molecular Aspects of Blood Cell–Endothelium Interactions in Vascular Disorders

In physiology and pathophysiology the molecules involved in blood cell–blood cell and blood cell–endothelium interactions have been identified. Platelet aggregation and adhesion to the walls belonging to vessels involve glycoproteins (GP), GP llb and GP llla and the GP Ib–IX–V complex. Red blood cells (RBCs) in normal situations have little interaction with the endothelium. Abnormal adhesion of RBCs was first observed in sickle cell anemia involving vascular cell adhesion molecule (VCAM)-1, α4β1, Lu/BCAM, and intercellular adhesion molecule (ICAM)-4. More recently RBC adhesion was found to be increased in retinal-vein occlusion (RVO) and in polycythemia vera (PV). The molecules which participate in this process are phosphatidylserine and annexin V in RVO, and phosphorylated Lu/BCAM and α5 laminin chain in PV. The additional adhesion in diabetes mellitus occurs due to the glycated RBC band 3 and the advanced glycation end-product receptors. The multiligand receptor binds advanced glycation end products (AGEs) or S100 calgranulins, or β-amyloid peptide. This receptor for advanced glycation end products is known as RAGE. The binding to RAGE-activated endothelial cells leads to an inflammatory reaction and a prothrombotic state via NADPH activation and altered gene expression. RAGE blockade is a potential target for drugs preventing the deleterious consequences of RAGE activation.

Comparison of intermittent fasting and exercise on skeletal muscle autophagy activation and fat reduction

To observe the effect of weight control of intermittent fasting in different time course (14 d, 28 d)and explore its effect on skeletal muscle mass and autophagy. Sixty SD rats (male) were randomly divided into sedentary group (Sed), intermittent fasting group (InF) and exercise group (Exe), 20 rats of each group, intervention duration is 14 d and 28 d. Animals of InF group were fasted every other day, animals of Exe group underwent aerobic exercise on treadmill, the weight was recorded every week. The body fat mass was recorded by DEXA and then the fat mass index was calculated. The wet weight of bilateral soleus muscle was weighed and wet weight index was calculated. Immunofluorescence was used to detect laminin and light chain 3 (LC3), which reflect muscle fiber cross-sectional area and autophagosome respectively. Transmission electron microscopy was used to observe the number and morphology of autophagosomes. The expression levels of autophagy related proteins Unc-51 like kinase 1 (ULK1), LC3, sequestosome1 protein (p62), AMP activated protein kinase (AMPKα) and p-AMPKα (Thr172) were detected by Western blot. ① From the 7th day of intervention, the body weight of rats in InF and Exe groups was significantly lower than that in Sed group, and the body weight of InF group was significantly lower than that in Exe group (P＜0.01). After 28 days of intervention, the fat mass index in InF and Exe groups was significantly lower than that in Sed group, the fat mass index of InF group was significantly lower than that in Exe group (P＜0.05).② After 28 days of intervention, the cross-sectional of muscle fibers in Exe group was significantly larger than that in Sed and InF groups (P＜0.01). ③The expressions of AMPKα, p-AMPKα (Thr172) and ULK1 in InF and Exe groups were significantly higher than those in Sed group (P＜0.05). However, at 14 days, only InF group showed the increase of LC3-II/LC3-I ratio and the decrease of p62 level (P＜0.05). The same indicators of Exe group were only significant changed at 28 d. ① Intermittent fasting is superior to exercise in controlling the growth of body weight and body fat in rats. ② In terms of skeletal muscle autophagy activation, the length of intervention required for intermittent fasting (14 days) is shorter than aerobic exercise (28 days).

Knockdown of the α5 laminin chain affects differentiation of colorectal cancer cells and their sensitivity to chemotherapy

The interaction of tumor cells with the extracellular matrix (ECM) may affect the rate of cancer progression and metastasis. One of the major components of ECM are laminins, the heterotrimeric glycoproteins consisting of α-, β-, and γ-chains (αβγ). Laminins interact with their cell surface receptors and, thus, regulate multiple cellular processes. In this work, we demonstrate that shRNA-mediated knockdown of the α5 laminin chain results in Wnt- and mTORC1-dependent partial dedifferentiation of colorectal cancer cells. Furthermore, we showed that this dedifferentiation involved activation of ER-stress signaling, pathway promoting the sensitivity of cells to 5-fluorouracil.

Bioactive fragments of laminin and collagen chains: lesson from the testis.

Recent studies have shown that the testis is producing several biologically active peptides, namely the F5- and the NC1-peptides from laminin-γ3 and collagen α3 (IV) chain, respectively, that promotes blood-testis barrier (BTB) remodeling and also elongated spermatid release at spermiation. Also the LG3/4/5 peptide from laminin-α2 chain promotes BTB integrity which is likely being used for the assembly of a 'new' BTB behind preleptotene spermatocytes under transport at the immunological barrier. These findings thus provide a new opportunity for investigators to better understand the biology of spermatogenesis. Herein, we briefly summarize the recent findings and provide a critical update. We also present a hypothetical model which could serve as the framework for studies in the years to come.

Recurrent urinary retention: An unusual presentation of laryngo-onycho-cutaneous syndrome

Laryngo-onycho-cutaneous (LOC) first described by Shabbir in 1986 in Muslim families from Punjab is a rare autosomal recessive disorder characterized by increased skin and mucous membrane fragility associated with exuberant granulation. The 4th International Consensus Meeting on “Diagnosis and Classification of Epidermolysis Bullosa” classifies it as a localized form of Junctional Epidermolysis Bullosa (JEB-LOC Syndrome) with mutations in α3 chain of Laminin 332. While laryngeal, ocular, and cutaneous involvement in these cases has been widely reported, urinary tract involvement is relatively rare. We report a case of JEB-LOC syndrome in a child from South India with extensive urethral involvement.

Laminin α4 contributes to airway remodeling and inflammation in asthma.

Airway inflammation and remodeling are characteristic features of asthma, with both contributing to airway hyperresponsiveness (AHR) and lung function limitation. Airway smooth muscle (ASM) accumulation and extracellular matrix deposition are characteristic features of airway remodeling, which may contribute to persistent AHR. Laminins containing the α2-chain contribute to characteristics of ASM remodeling in vitro and AHR in animal models of asthma. The role of other laminin chains, including the laminin α4 and α5 chains, which contribute to leukocyte migration in other diseases, is currently unknown. The aim of the current study was to investigate the role of these laminin chains in ASM function and in AHR, remodeling, and inflammation in asthma. Expression of both laminin α4 and α5 was observed in the human and mouse ASM bundle. In vitro, laminin α4 was found to promote a pro-proliferative, pro-contractile, and pro-fibrotic ASM cell phenotype. In line with this, treatment with laminin α4 and α5 function-blocking antibodies reduced allergen-induced increases in ASM mass in a mouse model of allergen-induced asthma. Moreover, eosinophilic inflammation was reduced by the laminin α4 function-blocking antibody as well. Using airway biopsies from healthy subjects and asthmatic patients, we found inverse correlations between ASM α4-chain expression and lung function and AHR, whereas eosinophil numbers correlated positively with expression of laminin α4 in the ASM bundle. This study, for the first time, indicates a prominent role for laminin α4 in ASM function and in inflammation, AHR, and remodeling in asthma, whereas the role of laminin α5 is more subtle.

068 Serological characterization of anti-p200 pemphigoid: Epitope spreading as a common phenomenon

Anti-p200 pemphigoid is an orphan subepidermal blistering disease characterized by autoantibody binding to the dermal site of human salt-split skin (SSS) and reactivity against a 200 kDa protein in the extract of human dermis by immunoblotting. Here, we aimed at the thorough serological characterization of anti-p200 pemphigoid. This retrospective study included all 243 patients with anti-p200 pemphigoid diagnosed between 2008 and 2017 in our routine autoimmune laboratory. Patients were diagnosed based on a compatible clinical picture and reactivity with the p200 protein in dermal extract by immunoblotting as described previously. In addition, all sera were subjected to indirect immunofluorescence microscopy on SSS and immunoblotting with the extracellular matrix of cultured keratinocytes for reactivity against laminin 332 and ELISA or immunoblotting applying the recombinant NC1 domain of collagen type VII. Autoantibodies against the dermal side of SSS were found in 91% (221/243) of patients and against the p200 protein and laminin γ1 in 100% (243/243) and 88% (209/238) of sera, respectively. IgG reactivity to laminin 332 was observed in 21% (48/233) of patients, in 98% of those (47/48) against the α3 chain of laminin 332. IgG autoantibodies to type VII collagen were detected in 2% (4/243) of patients. 30% (73/243) of sera showed additional reactivity to the epidermal site of SSS. In 58% (42/73) of epidermal binding sera, antibodies against BP180 or BP230 were detected by ELISA, while in 42% (31/73) of these sera, the target antigen remained unclear. In 37% (89/243) of sera, reactivity against one antigen in addition to p200/laminin γ1 and in 7% (16/243) against 2 additional antigens was detected. In summary, in this so far largest cohort of anti-p200 pemphigoid patients, epitope spreading was a common phenomenon and most frequently included laminin 332 followed by BP180, BP230, and type VII collagen.

Tumour-Derived Laminin α5 (LAMA5) Promotes Colorectal Liver Metastasis Growth, Branching Angiogenesis and Notch Pathway Inhibition.

Hepatic metastatic growth is dependent upon stromal factors including the matrisomal proteins that make up the extracellular matrix (ECM). Laminins are ECM glycoproteins with several functions relevant to tumour progression including angiogenesis. We investigated whether metastatic colon cancer cells produce the laminins required for vascular basement membrane assembly as a mechanism for the promotion of angiogenesis and liver metastasis growth. qPCR was performed using human-specific primers to laminin chains on RNA from orthotopic human colorectal liver metastases. Laminin α5 (LAMA5) expression was inhibited in colon cancer cells using shRNA. Notch pathway gene expression was determined in endothelia from hepatic metastases. Orthotopic hepatic metastases expressed human laminin chains α5, β1 and γ1 (laminin 511), all of which are required for vascular basement membrane assembly. The expression of Laminin 511 was associated with reduced survival in several independent colorectal cancer cohorts and angiogenesis signatures or vessel density significantly correlated with LAMA5 expression. Colorectal cancer cells in culture made little LAMA5, but its levels were increased by culture in a medium conditioned by tumour-derived CD11b+ myeloid cells through TNFα/NFκB pathway signalling. Down-regulation of LAMA5 in cancer cells impaired liver metastatic growth and resulted in reduced intra-tumoural vessel branching and increased the expression of Notch pathway genes in metastasis-derived endothelia. This data demonstrates a mechanism whereby tumour inflammation induces LAMA5 expression in colorectal cancer cells. LAMA5 is required for the successful growth of hepatic metastases where it promotes branching angiogenesis and modulates Notch signalling.

RNA-dependent Amplification of Mammalian mRNA Encoding Extracellullar Matrix Proteins: Identification of Chimeric RNA Intermediates for α1, β1, and γ1 Chains of Laminin

De novo production of RNA on RNA template, a process known as RNA-dependent RNA synthesis, RdRs, and the enzymatic activity conducting it, RNA-dependent RNA polymerase, RdRp, were initially considered to be exclusively virus-specific. Eventually, however, the occurrence of RdRs and the ubiquitous presence of conventional RdRp were demonstrated in numerous eukaryotic organisms. The evidence that the enzymatic machinery capable of RdRs is present in mammalian cells was derived from studies of viruses, such as hepatitis delta virus, HDV, that do not encode RdRp yet undergo a robust RNA replication once inside the mammalian host; thus firmly establishing its occurrence and functionality. Moreover, it became clear that RdRp activity, apparently in a non-conventional form, is constitutively present in most, if not in all, mammalian cells. Because such activity was shown to produce short transcripts, given its apparent involvement in RNA interference phenomena, and because double-stranded RNA is known to trigger cellular responses leading to its degradation, it was generally assumed that its role in mammalian cells is restricted to a regulatory function. However, at the same time, an enzymatic activity capable of generating complete antisense RNA complements of mRNAs was discovered in mammalian cells undergoing terminal differentiation. Moreover, observations of widespread synthesis of antisense RNAs initiating at the 3'poly(A) of mRNAs in human cells suggested an extensive cellular utilization of mammalian RdRp. These results led to the development of a model of RdRp-facilitated and antisense RNA-mediated amplification of mammalian mRNA. Recent detection of the major model-predicted identifiers, chimeric RNA intermediates containing both sense and antisense RNA strands covalently joined in a rigorously predicted and uniquely defined manner, as well as the identification of a putative chimeric RNA end product of this process, validated the proposed model. The results corroborating mammalian RNA-dependent mRNA amplification were obtained in vivo with cells undergoing terminal erythroid differentiation and programmed for only a short survival span. This raises a question of whether mammalian RNA-dependent mRNA amplification is a specialized occurrence limited to extreme circumstances of terminal differentiation or a general physiological phenomenon. The present study addresses this question by testing for the occurrence of RNA-dependent amplification of mRNA encoding extracellular matrix proteins abundantly produced throughout the tissue and organ development and homeostasis, an exceptionally revealing indicator of the range and scope of this phenomenon. We report here the detection of major identifiers of RNA-dependent amplification of mRNA encoding α1, β1, and γ1 chains of laminin in mouse tissues producing large quantities of extracellular matrix proteins. The results obtained warrant reinterpretation of the mechanisms involved in ubiquitous and abundant production and deposition of extracellular matrix proteins, confirm the occurrence of mammalian RNA-dependent mRNA amplification as a new mode of genomic protein-encoding information transfer, and establish it as a general physiological phenomenon.

Protein-Encoding RNA to RNA Information Transfer in Mammalian Cells: RNA-dependent mRNA Amplification. Identification of Chimeric RNA Intermediates and Putative RNA End Products

Our initial unidirectional understanding of the flow of protein-encoding genetic information, DNA to RNA to protein, a process defined as the "Central Dogma of Molecular Biology" and usually depicted as a downward arrow, was eventually amended to account for the "vertical" information back-flow from RNA to DNA, reverse transcription, and for its "horizontal" side-flow from RNA to RNA, RNA-dependent RNA synthesis, RdRs. These processes, both potentially leading to protein production, were assumed to be strictly virus-specific. However, whereas this presumption might be true for the former, it became apparent that the cellular enzymatic machinery for the later, a conventional RNA-dependent RNA polymerase activity, RdRp, is ubiquitously present and RdRs regularly occurs in eukaryotes. The strongest evidence for the occurrence and functionality of RdRp activity in mammalian cells comes from viruses, such as hepatitis delta virus, HDV, that do not encode RdRp yet undergo a robust RNA replication once inside the host. Eventually, it became clear that RdRp activity, apparently in a non-conventional form, is constitutively present in most, if not in all, mammalian cells. Because such activity was shown to produce short transcripts, because of its apparent involvement in RNA interference phenomena, and because double-stranded RNA is known to trigger cellular responses leading to its degradation, it was generally assumed that its role in mammalian cells is restricted to a regulatory function. However, at the same time, an enzymatic activity capable of generating complete antisense RNA complements of mRNAs was discovered in mammalian cells undergoing terminal differentiation. Moreover, observations of widespread synthesis of antisense RNA initiating at the 3'poly(A) of mRNAs in human cells suggested an extensive cellular utilization of mammalian RdRp. These results led to the development of a model of RdRp-facilitated and antisense RNA-mediated amplification of mammalian mRNA. Here, we report the in vivo detection in cells undergoing terminal erythroid differentiation of the major model-predicted identifiers of such a process, a chimeric double-stranded/pinhead-structured intermediates containing both sense and antisense RNA strands covalently joined in a rigorously predicted and uniquely defined manner. We also report the identification of the putative chimeric RNA end product of mRNA amplification. It is heavily modified, uniformly truncated, yet retains the intact coding region, and terminates with the OH group at both ends; its massive cellular amount is unprecedented for a conventional mRNA transcription product and it translates into polypeptides indistinguishable from the translation product of conventional mRNA. Moreover, we describe the occurrence of the second Tier of mammalian RNA-dependent mRNA amplification, a physiologically occurring, RdRp-driven intracellular PCR process, "iPCR", and report the detection of its distinct RNA end products. Whether mammalian mRNA amplification is a specialized occurrence limited to extreme circumstances of terminal differentiation in cells programmed for only a short survival span or a general physiological phenomenon was answered in the companion article Volloch et al. Ann Integr Mol Med. 2019;1(1):1004. by the detection of major identifiers of this process for mRNA encoding α1, β1, and γ1 chains of laminin, a major extracellular matrix protein abundantly produced throughout the tissue and organ development and homeostasis and an exceptionally revealing indicator of the range and scope of this phenomenon. The results obtained introduce the occurrence of RNA-dependent mRNA amplification as a new mode of genomic protein-encoding information transfer in mammalian cells and establish it as a general physiological phenomenon.