Neuronal Phenotype of col4a1 and col25a1: An Intriguing Hypothesis in Vertebrates Brain Aging.

Collagens are the most abundant proteins in vertebrates and constitute the major components of the extracellular matrix. Collagens play an important and multifaceted role in the development and functioning of the nervous system and undergo structural remodeling and quantitative modifications during aging. Here, we investigated the age-dependent regulation of col4a1 and col25a1 in the brain of the short-lived vertebrate Nothobranchius furzeri, a powerful model organism for aging research due to its natural fast-aging process and further characterized typical hallmarks of brain aging in this species. We showed that col4a1 and col25a1 are relatively well conserved during vertebrate evolution, and their expression significantly increases in the brain of N. furzeri upon aging. Noteworthy, we report that both col4a1 and col25a1 are expressed in cells with a neuronal phenotype, unlike what has already been documented in mammalian brain, in which only col25a1 is considered a neuronal marker, whereas col4a1 seems to be expressed only in endothelial cells. Overall, our findings encourage further investigation on the role of col4a1 and col25a1 in the biology of the vertebrate brain as well as the onset of aging and neurodegenerative diseases.

NC1-peptide derived from collagen α3 (IV) chain is a blood-tissue barrier regulator: lesson from the testis.

Collagen α3 (IV) chains are one of the major constituent components of the basement membrane in the mammalian testis. Studies have shown that biologically active fragments, such as noncollagenase domain (NC1)-peptide, can be released from the C-terminal region of collagen α3 (IV) chains, possibly through the proteolytic action of metalloproteinase 9 (MMP9). NC1-peptide was shown to promote blood-testis barrier (BTB) remodeling and fully developed spermatid (e.g., sperm) release from the seminiferous epithelium because this bioactive peptide was capable of perturbing the organization of both actin- and microtubule (MT)-based cytoskeletons at the Sertoli cell-cell and also Sertoli-spermatid interface, the ultrastructure known as the basal ectoplasmic specialization (ES) and apical ES, respectively. More importantly, recent studies have shown that this NC1-peptide-induced effects on cytoskeletal organization in the testis are mediated through an activation of mammalian target of rapamycin complex 1/ribosomal protein S6/transforming retrovirus Akt1/2 protein (mTORC1/rpS6/Akt1/2) signaling cascade, involving an activation of cell division control protein 42 homolog (Cdc42) GTPase, but not Ras homolog family member A GTPase (RhoA), and the participation of end-binding protein 1 (EB1), a microtubule plus (+) end tracking protein (+TIP), downstream. Herein, we critically evaluate these findings, providing a critical discussion by which the basement membrane modulates spermatogenesis through one of its locally generated regulatory peptides in the testis.

NC1-Peptide From Collagen α3 (IV) Chains in the Basement Membrane of Testes Regulates Spermatogenesis via p-FAK-Y407.

The blood-testis barrier (BTB) in the testis is an important ultrastructure to support spermatogenesis. This blood-tissue barrier undergoes remodeling at late stage VII to early stage IX of the epithelial cycle to support the transport of preleptotene spermatocytes across the BTB to prepare for meiosis I/II at the apical compartment through a mechanism that remains to be delineated. Studies have shown that NC1-peptide-derived collagen α3 (IV) chain in the basement membrane is a bioactive peptide that induces BTB remodeling. It also promotes the release of fully developed spermatids into the tubule lumen. Thus, this endogenously produced peptide coordinates these 2 cellular events across the seminiferous epithelium. Using an NC1-peptide complementary deoxyribonucleic acid (cDNA) construct to transfect adult rat testes for overexpression, NC1-peptide was found to effectively induce germ cell exfoliation and BTB remodeling, which was associated with a surge and activation of p-rpS6, the downstream signaling protein of mTORC1 and the concomitant downregulation of p-FAK-Y407 in the testis. In order to define the functional relationship between p-rpS6 and p-FAK-Y407 signaling to confer the ability of NC1-peptide to regulate testis function, a phosphomimetic (and thus constitutively active) mutant of p-FAK-Y407 (p-FAK-Y407E-MT) was used for its co-transfection, utilizing Sertoli cells cultured in vitro with a functional tight junction (TJ) barrier that mimicked the BTB in vivo. Overexpression of p-FAK-Y407E-MT blocked the effects of NC1-peptide to perturb Sertoli cell BTB function by promoting F-actin and microtubule cytoskeleton function, and downregulated the NC1-peptide-mediated induction of p-rpS6 activation. In brief, NC1-peptide is an important endogenously produced biomolecule that regulates BTB dynamics.

Expanding the phenotype of COL4A1-related disorders-Four novel variants.

OBJECTIVE: COL4A1 variant causes severe central nervous system (CNS) anomalies, including hydranencephaly. However, the pathogenic mechanism underlying the COL4A1 phenotype remains unclear. Here, we report de novo COL4A1 variants in four Japanese patients with typical or rare CNS involvement and exhibiting diverse phenotypes. METHODS: We identified and enrolled four patients with white matter abnormalities and cerebral structural defects suggestive of cerebrovascular disease. Genetic analysis was performed using panel sequencing. RESULTS: All the patients were perinatally asymptomatic during the infantile period but exhibited developmental delay and growth retardation later. All the patients exhibited CNS symptoms, including psychomotor disability, spastic paralysis, and epilepsy. Brain magnetic resonance imaging revealed hydranencephaly (n = 1), ventriculomegaly (n = 4) associated with cerebral hemorrhage, and atretic encephalocele (n = 1). Three patients had developed congenital cataract, while two had hematuria. We identified two COL4A1 missense variants [exon32:c.2555G > A p.(Gly852Asp), exon40:c.3407G > A p.(Gly1136Asp)] and two in frame variants [exon32:c.2603_2609delinsATCCTGA p.(Ala868_Gly870delinsAspProGlu), exon36:c.3054delinsTGTAGAT p.(Leu1018delinsPheValAsp)]. The in frame variants were associated with severe CNS anomalies, hydranencephaly, and severe ventriculomegaly. Atretic encephalocele has never been reported in individuals with COL4A1 variants. CONCLUSIONS: Our findings suggest that COL4A1 variants cause variable CNS symptoms. Association between clinical phenotypes and each COL4A1 variant would clarify their underlying etiologies.

Angiotensin-[1-7] attenuates kidney injury in experimental Alport syndrome.

Angiotensin-[1-7] (Ang-[1-7]) antagonize the actions of the renin-angiotensin-system via the Mas receptor and thereby exert renoprotective effects. Murine recombinant angiotensin-converting enzyme (ACE)2 was reported to show renoprotective effects in an experimental Alport syndrome model; however, the protective effect of direct administration of Ang-[1-7] is unknown. Here, we used Col4a3-/- mice as a model of Alport syndrome, which were treated with saline or Ang- [1-7]; saline-treated wild-type mice were used as a control group. The mice were continuously infused with saline or Ang-[1-7] (25 µg/kg/h) using osmotic mini-pumps. Col4a3-/- mice showed increased α-smooth muscle actin (SMA), collagen, and fibronectin expression levels, which were attenuated by Ang-[1-7] treatment. Moreover, Ang-[1-7] alleviated activation of transforming growth factor-ß/Smad signaling, and attenuated the protein expression of ED-1 and heme oxygenase-1, indicating reduction of renal inflammation. Ang-[1-7] treatment further reduced the expression levels of inflammatory cytokines and adhesion molecules and attenuated apoptosis in human kidney cells. Finally, Ang-[1-7] downregulated TNF-α converting enzyme and upregulated ACE2 expression. Thus, treatment with Ang-[1-7] altered the ACE2-Ang-[1-7]-Mas receptor axis in the kidneys of Col4a3-/- mice to attenuate the nephropathy progression of Alport syndrome.

Pericardin, a Drosophila collagen, facilitates accumulation of hemocytes at the heart.

Hematopoietic cell lineages support organismal needs by responding to positional and systemic signals that balance proliferative and differentiation events. Drosophila provides an excellent genetic model to dissect these signals, where the activity of cues in the hemolymph or substrate can be traced to determination and differentiation events of well characterized hemocyte types. Plasmatocytes in third instar larvae increase in number in response to infection and in anticipation of metamorphosis. Here we characterize hemocyte clustering, proliferation and transdifferentiation on the heart or dorsal vessel. Hemocytes accumulate on the inner foldings of the heart basement membrane, where they move with heart contraction, and are in proximity to the heart ostia and pericardial nephrocytes. The numbers of hemocytes vary, but increase transiently before pupariation, and decrease by 4 h before pupa formation. During their accumulation at the heart, plasmatocytes can proliferate and can transdifferentiate into crystal cells. Serrate expressing cells as well as lamellocyte-like, Atilla expressing ensheathing cells are associated with some, but not all hemocyte clusters. Hemocyte aggregation is enhanced by the presence of a heart specific Collagen, Pericardin, but not the associated pericardial cells. The varied and transient number of hemocytes in the pericardial compartment suggests that this is not a hematopoietic hub, but a niche supporting differentiation and rapid dispersal in response to systemic signals.

Inflammation by activated macrophage-like THP-1 cells increases human dura mater cell adhesion with alteration of integrin α2 ß1 and matrix metalloproteinase.

This study was designed to investigate (i) extracellular matrix to specify adhesive substrates to human dura mater cell (hDMC); (ii) the alteration on adhesion-related molecules in hDMC; and (iii) secreted matrix metalloproteinases (MMPs) linked with extracellular matrix remodeling after exposure to inflammation. The hDMC was cultured from human dura mater tissue, and the studies were performed with hDMC after co-culturing with macrophage like THP-1 cells (Mϕ). The adhesion of co-cultured hDMC through collagen I increased 6.4-fold and through collagen IV increased 5.0-fold compared with the adhesion of naïve cells (p < 0.001). Integrin subtype α2 ß1 expression was increased 6.3-fold (p < 0.001) and α1 expression was decreased 2.0-fold (p < 0.001) in the co-cultured cells compared with the naïve cells. Co-culturing induced significant increases in MMP-1 (13.9-fold, p < 0.01), MMP-3 (7.6-fold, p < 0.01), and VEGF (VEGF: 3.8-fold, p < 0.05) expression and decreases in MMP-9 (0.1-fold, p < 0.01) compared with the sum of naïve hDMC and Mϕ values. Increased hDMC adhesion under inflammatory conditions is caused by an increased cellular affinity for collagen I and IV mediated by increased hDMC levels of integrin subtype α2 ß1 and environmental MMP-1, -3 and decreased MMP-9. Selective integrin subtype α2 ß1 inhibition assay showed 37.8% and 35.7% reduction in adhesion of co-cultured hDMC to collagen I (p < 0.001) and IV (p = 0.057), respectively. The present study provides insight into the pathological conditions related to dura mater adhesion in inflammation. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-11, 2019.

Effect of Mesenchymal Cells on Myoblast Sheets Embedded in Collagen Gel.

The objective of this study was to investigate the effects of mesenchymal cells on myoblasts in long-term cultivation of myoblast cell sheets. Sheets of myoblasts and mesenchymal cells from Japanese rabbit oral mucosa were generated and analyzed by histochemistry, Western blot, and reverse transcription-polymerase chain reaction. The presence of desmin and type IV collagen, which is seen in normal muscle tissue, was also confirmed in all the sheets produced. Expression of desmin and type IV collagen showed a decrease under co-culture conditions. In addition, expression of genes important in maintaining the undifferentiated state (Pax7, CD34, myogenin, MyoD) in myoblasts was observed throughout the long cultivation period. Insulin-like growth factor was expressed only when the mesenchymal cells were co-cultured with myoblasts. These data suggest that the presence of mesenchymal cells in a long-term co-culture system influences myoblast differentiation.

Canstatin modulates L-type calcium channel activity in rat ventricular cardiomyocytes.

Excessive increase of cytosolic Ca2+ through the activation of L-type Ca2+ channels (LTCCs) via ß adrenergic receptor induces apoptosis of cardiomyocytes. Canstatin, a cleaved fragment of collagen type IV α2 chain, is abundantly expressed in normal heart tissue. We previously reported that canstatin inhibits ß adrenergic receptor-stimulated apoptosis in cardiomyoblasts. Here, we tested the hypothesis that canstatin regulates LTCCs activity in ventricular cardiomyocytes. Collagen type IV α2 chain (COL4A2) small interfering (si) RNA (for canstatin suppression) or control siRNA was injected via jugular vein in Wistar rats. Two days after the injection, electrocardiogram (ECG) was recorded and the left ventricular tissue was isolated using Langendorff apparatus. Immunofluorescence staining was performed to clarify the distribution of canstatin in cardiomyocytes. The knockdown efficiency was confirmed by Western blotting. The L-type Ca2+ channel current (ICaL) of ventricular cardiomyocyte was measured by a whole-cell patch clamp technique. In immunofluorescence staining, colocalization of canstatin and αv integrin was observed in the isolated ventricular cardiomyocytes. The ICaL of ventricular cardiomyocyte isolated from COL4A2 siRNA-injected rats was significantly enhanced compared with control siRNA-injected rats. Recombinant canstatin (250 ng/ml) significantly reversed it. ECG analysis showed that QT interval tended to be shortened and amplitude of T wave was significantly increased in the COL4A2 siRNA-injected rats. In summary, we for the first time clarified that suppressing canstatin expression increases the basal ICaL in ventricular cardiomyocytes. It is proposed that canstatin might play a role in the stabilization of cardiac function through the modulation of LTCC activity in cardiomyocytes.

Type IV collagen α6 chain is a regulator of keratin 10 in keratinization of oral mucosal epithelium.

Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue, and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva. Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells. Therefore, first, to identify the keratinized mucosa-specific basement membrane components, immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice. No difference in the expression pattern of type IV collagen α1(IV) and α2(IV) chains was observed in the keratinized and non-keratinized mucosa. Interestingly, however, type IV collagen α5(IV) and α6(IV) chains specifically were strongly detected in the keratinized mucosa. To analyze the functional roles of the type IV collagen isoform α6(IV) in oral mucosa keratinization, we analyzed Col4a6-knockout mice. Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice. Additionally, in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of α6(IV) chain in epithelial keratinization. These findings indicate that α112:α556 (IV) network, which is the only network that includes the α6(IV) chain, is one regulator of KRT10 expression in keratinization of oral mucosal epithelium.

Basement membrane collagen IV: Isolation of functional domains.

Collagen IV is a major constituent of basement membranes, specialized form of extracellular matrix that provides a mechanical support for tissues, serves as a polyvalent ligand for cell adhesion receptors and as a scaffold for other proteins, and plays a key role in tissue genesis, differentiation, homeostasis, and remodeling. Collagen IV underlies the pathogenesis of several human disorders including Goodpasture's disease, Alport's syndrome, diabetic nephropathy, angiopathy, and porencephaly. While the isolation of the collagen IV molecules from tissues is an ultimate prerequisite for structural and functional studies, it has been always hampered by the protein insolubility due to extensive intermolecular crosslinking and noncovalent associations with other components of basement membranes. In this chapter, we present methods for the isolation of collagen IV fragments from basement membranes or from extracellular matrix deposited by cultured cells, and the recombinant expression alternative. These methods are useful to address the fundamental questions on the role of collagen IV in tissue genesis under the normal and pathological conditions.

Type IV collagen aggregates promote keratinocyte proliferation and formation of epidermal layer in human skin equivalents.

Type IV collagen isolated from lens capsule without enzymatic treatment is known to form a gel under physiological condition and influences cellular activities. In case of human keratinocytes, the suppression of proliferation on reconstituted type IV collagen gels was reported in monolayer culture. In this study, we examined effects of type IV collagen isolated from porcine lens capsule on epidermal formation in human skin equivalents (HSEs). Type IV collagen aggregates were prepared under the culture condition and the aggregates suppressed keratinocyte proliferation in monolayer culture as well as the culture on the gels. In HSEs, type IV collagen aggregates were reconstituted on the surface of contracted collagen gels containing human dermal fibroblasts and the keratinocytes were then cultured on the aggregates for 14 days. Interestingly, in HSEs with type IV collagen aggregates, the BrdU-positive keratinocytes were increased and the thickness of the epidermal layer was around twice than that of control culture. Epidermal differentiation markers were expressed in the upper layer of the epidermis and the defined deposition of human basement membrane components were increased at the dermal-epidermal junction. These results indicate that the type IV collagen aggregates stimulate the proliferation of basal keratinocytes and improve the stratification of epidermal layers in HSEs.

Abdominal wall hernias-A local manifestation of systemically impaired quality of the extracellular matrix.

BACKGROUND: Throughout life, inguinal hernia develops in approximately every fourth man, some of whom develop multiple hernias. If patients at risk of developing multiple hernias could be identified by a serologic biomarker, treatment might be able to be tailored and improved. Evidence suggests that abdominal wall hernia formation is associated with altered collagen metabolism. The aim of this study was to evaluate biomarkers for type IV and V collagen turnover in patients with multiple hernias and control subjects without hernia. METHODS: Venous blood was collected from 88 men (mean age, 62 years) with a history of more than 3 hernia repairs and 86, age-matched men without hernias. Biomarkers for synthesis of collagen type IV (P4NP) and type V (P5CP) as well as breakdown (C4M and C5M) were measured in serum by validated, solid-phase, competitive assays. Collagen turnover was indicated by the ratio between the biomarker for synthesis and breakdown. RESULTS: Type IV collagen turnover was 1.4-fold increased in patients with multiple hernias compared to control subjects (P < .001), whereas type V collagen turnover was 1.7-fold decreased (P < .001). Diagnostic power of P5CP was 0.83 (95%C.I.:0.77-0.89), P < .001. CONCLUSION: Patients with multiple hernias exhibit increased turnover of type IV collagen and a decreased turnover of type V collagen, demonstrating systemically altered collagen turnover. Biomarkers for type V collagen turnover may be used to identify patients at risk for or with multiple hernias.

Constructing bio-layer of heparin and type IV collagen on titanium surface for improving its endothelialization and blood compatibility.

The modification of cardiovascular stent surface for a better micro-environment has gradually changed to multi-molecule, multi-functional designation. In this study, heparin (Hep) and type IV collagen (IVCol) were used as the functional molecule to construct a bifunctional micro-environment of anticoagulation and promoting endothelialization on titanium (Ti). The surface characterization results (AFM, Alcian Blue 8GX Staining and fluorescence staining of IVCol) indicated that the bio-layer of Hep and IVCol were successfully fabricated on the Ti surface through electrostatic self-assembly. The APTT and platelet adhesion test demonstrated that the bionic layer possessed better blood compatibility compared with Ti surface. The adhesion, proliferation, migration and apoptosis tests of endothelial cells proved that the Hep/IVCol layer was able to enhance the endothelialization of the Ti surface. The in vivo animal implantation results manifested that the bionic surface could encourage new endothelialization. This work provides an important reference for the construction of multifunction micro-environment on the cardiovascular scaffold surface.

Concomitant neoplasms in the skin and stomach unveil the role of type IV collagen and E-cadherin in mucin core protein 5AC expression in vivo.

Mucin core protein (MUC) 5AC is a gel-forming glycoprotein that is expressed in different types of tumour cells. MUC5AC expression in cultured cells is regulated through the extracellular matrix and through remodelling by other membranous proteins such as type IV collagen (COL4) and E-cadherin. However, it has not been elucidated whether COL4 and E-cadherin affect MUC5AC expression in tumours in vivo. Here, by analysing a single individual with concomitant neoplasms in the skin [extramammary Paget disease (EMPD)] and the stomach (gastric cancer), we show that MUC5AC expression is reduced in COL4 and membranous E-cadherin-expressing EMPD specimens whereas MUC5AC is not abolished in gastric cancer with COL4 negativity and E-cadherin cytoplasmic localization. As the EMPD and gastric cancer specimens were derived from a single patient, each specimen had the same genetic background. These in vivo results support previous in vitro studies which showed that COL4 and E-cadherin downregulated MUC5AC expression. Our study suggests that concomitant neoplasms in different organs of the same individual can serve as a strong tool for uncovering functional diversity in tumour markers in distinct cancer cells.

Finite element implementation of a multiscale model of the human lens capsule.

An axisymmetric finite element implementation of a previously described structural constitutive model for the human lens capsule (Burd in Biomech Model Mechanobiol 8(3):217-231, 2009) is presented. This constitutive model is based on a hyperelastic approach in which the network of collagen IV within the capsule is represented by an irregular hexagonal planar network of hyperelastic bars, embedded in a hyperelastic matrix. The paper gives a detailed specification of the model and the periodic boundary conditions adopted for the network component. Momentum balance equations for the network are derived in variational form. These balance equations are used to develop a nonlinear solution scheme to enable the equilibrium configuration of the network to be computed. The constitutive model is implemented within a macroscopic finite element framework to give a multiscale model of the lens capsule. The possibility of capsule wrinkling is included in the formulation. To achieve this implementation, values of the first and second derivatives of the strain energy density with respect to the in-plane stretch ratios need to be computed at the local, constitutive model, level. Procedures to determine these strain energy derivatives at equilibrium configurations of the network are described. The multiscale model is calibrated against previously published experimental data on isolated inflation and uniaxial stretching of ex vivo human capsule samples. Two independent example lens capsule inflation analyses are presented.

Molecular and Genetic Analyses of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention.

BACKGROUND: Collagen type IV alpha1 (COL4A1) and alpha2 (COL4A2) form heterotrimers critical for vascular basement membrane stability and function. Patients with COL4A1 or COL4A2 mutations suffer from diverse cerebrovascular diseases, including cerebral microbleeds, porencephaly, and fatal intracerebral hemorrhage (ICH). However, the pathogenic mechanisms remain unknown, and there is a lack of effective treatment. METHODS AND RESULTS: Using Col4a1 and Col4a2 mutant mouse models, we investigated the genetic complexity and cellular mechanisms underlying the disease. We found that Col4a1 mutations cause abnormal vascular development, which triggers small-vessel disease, recurrent hemorrhagic strokes, and age-related macroangiopathy. We showed that allelic heterogeneity, genetic context, and environmental factors such as intense exercise or anticoagulant medication modulated disease severity and contributed to phenotypic heterogeneity. We found that intracellular accumulation of mutant collagen in vascular endothelial cells and pericytes was a key triggering factor of ICH. Finally, we showed that treatment of mutant mice with a US Food and Drug Administration-approved chemical chaperone resulted in a decreased collagen intracellular accumulation and a significant reduction in ICH severity. CONCLUSIONS: Our data are the first to show therapeutic prevention in vivo of ICH resulting from Col4a1 mutation and imply that a mechanism-based therapy promoting protein folding might also prevent ICH in patients with COL4A1 and COL4A2 mutations.

Identifying a novel role for X-prolyl aminopeptidase (Xpnpep) 2 in CrVI-induced adverse effects on germ cell nest breakdown and follicle development in rats.

Environmental exposure to endocrine-disrupting chemicals (EDCs) is one cause of premature ovarian failure (POF). Hexavalent chromium (CrVI) is a heavy metal EDC widely used in more than 50 industries, including chrome plating, welding, wood processing, and tanneries. Recent data from U.S. Environmental Protection Agency indicate increased levels of Cr in drinking water from several American cities, which potentially predispose residents to various health problems. Recently, we demonstrated that gestational exposure to CrVI caused POF in F1 offspring. The current study was performed to identify the molecular mechanism behind CrVI-induced POF. Pregnant rats were treated with 25 ppm of potassium dichromate from Gestational Day (GD) 9.5 to GD 14.5 through drinking water, and the fetuses were exposed to CrVI through transplacental transfer. Ovaries were removed from the fetuses or pups on Embryonic Day (ED) 15.5, ED 17.5, Postnatal Day (PND) 1, PND 4, or PND 25, and various analyses were performed. Results showed that gestational exposure to CrVI: 1) increased germ cell/oocyte apoptosis and advanced germ cell nest (GCN) breakdown; 2) increased X-prolyl aminopeptidase (Xpnpep) 2, a POF marker in humans, during GCN breakdown; 3) decreased Xpnpep2 during postnatal follicle development; and 4) increased colocalization of Xpnpep2 with Col3 and Col4. We also found that Xpnpep2 inversely regulated the expression of Col1, Col3, and Col4 in all the developmental stages studied. Thus, CrVI advanced GCN breakdown and increased follicle atresia in F1 female progeny by targeting Xpnpep2.

Serum with phospholipase A2 receptor autoantibodies interferes with podocyte adhesion to collagen.

BACKGROUND: The majority of sera from patients with primary membranous nephropathy have autoantibodies against the M-type phospholipase A2 receptor (PLA2R) which is expressed on human podocytes. The rabbit variant of PLA2R attaches to collagen type IV via the fibronectin type II domain, which is also present in the human variant of PLA2R. DESIGN: To assess whether the human PLA2R variant is also involved in attachment to collagen type IV, we conducted a cell adhesion assay on a collagen-coated surface using PLA2R-transfected and mock-transfected human embryonic kidney (HEK) cells. To test the hypothesis that sera from patients containing anti-PLA2R antibodies interfere with the adhesion of podocytes to collagen, we performed cell adhesion assays on a collagen type IV-coated surface using positive and negative serum samples from patients and cultured human podocytes in vitro expressing PLA2R. RESULTS: The HEK cell adhesion assay confirmed an enhanced attachment of PLA2R-transfected cells to collagen type IV. We confirmed diminished podocyte adhesion in the presence of serum with anti-PLA2R antibodies. The concentration of anti-PLA2R antibodies correlated with proteinuria and to the degree of diminished adhesion of podocytes. CONCLUSIONS: We demonstrated that serum of patients containing autoantibodies directed to PLA2R interferes with the ability of podocytes to attach to collagen type IV in vitro, providing evidence of a serum soluble pathogenic factor interfering with podocyte adhesion in membranous nephropathy.

Overexpression of tumstatin in genetically modified megakaryocytes changes the proangiogenic effect of platelets.

BACKGROUND: Thrombocytopenia is a common side effect of tumor chemotherapy, the main management approach to which is based on platelet (PLT) transfusion. However, PLTs, containing angiogenesis regulators, play a major role in boosting tumor growth and metastasis. The purpose of the study was to determine whether PLTs have the capacity to overexpress tumstatin by modified megakaryocyte (MK) and PLT precursors using lentivirus-mediated gene transfer, which might lead to alteration in proangiogenic effect of PLTs. STUDY DESIGN AND METHODS: CD34+ hematopoietic stem cells (HSCs) were transduced with recombinant lentivirus carrying tumstatin and induced to produce MKs and PLTs in the culture medium containing a cytokine cocktail. Flow cytometry and aggregation test were used to detect the generation and function of MKs and PLTs. Western blot analysis and confocal microscopy were applied to examine the expression and distribution of tumstatin in transgenic MKs and PLTs. Capillary tube formation of human umbilical vein endothelial cells (HUVECs) was used to evaluate the inhibitory effect of transgenic PLTs. RESULTS: CD34+ HSCs can be efficiently transduced with lentivirus vectors and successfully differentiated into MKs and PLTs. Large amounts of functional MKs and PLTs could be generated and had correct biologic characteristics. The tests demonstrated the feasibility of tumstatin expression in MKs and PLTs under control of the cytomegalovirus promoter, that thus tumstatin was stored in the α-granules of PLTs, and that the releasate of thrombin or A543 cell-stimulated transgenic PLTs obviously inhibited the growth of capillary tube network structures of HUVECs. CONCLUSION: Gene-modified CD34+ HSCs not only successfully differentiated into MKs and PLTs but also expressed tumstatin protein. Release of tumstatin in transgenic PLT granules led to antiangiogenic effect of PLTs.