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.

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.

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 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.

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.

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.

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.

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.

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.

Feasibility of repairing glomerular basement membrane defects in Alport syndrome.

Alport syndrome is a hereditary glomerular disease that leads to kidney failure. It is caused by mutations affecting one of three chains of the collagen α3α4α5(IV) heterotrimer, which forms the major collagen IV network of the glomerular basement membrane (GBM). In the absence of the α3α4α5(IV) network, the α1α1α2(IV) network substitutes, but it is insufficient to maintain normal kidney function. Inhibition of angiotensin-converting enzyme slows progression to kidney failure in patients with Alport syndrome but is not a cure. Restoration of the normal collagen α3α4α5(IV) network in the GBM, by either cell- or gene-based therapy, is an attractive and logical approach toward a cure, but whether or not the abnormal GBM can be repaired once it has formed and is functioning is unknown. Using a mouse model of Alport syndrome and an inducible transgene system, we found that secretion of α3α4α5(IV) heterotrimers by podocytes into a preformed, abnormal, filtering Alport GBM is effective at restoring the missing collagen IV network, slowing kidney disease progression, and extending life span. This proof-of-principle study demonstrates the plasticity of the mature GBM and validates the pursuit of therapeutic approaches aimed at normalizing the GBM to prolong kidney function.

Neonatal Fc receptor promotes immune complex-mediated glomerular disease.

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.

Evidence for activation of the unfolded protein response in collagen IV nephropathies.

Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)-related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies.

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.

Antifibrotic, nephroprotective effects of paricalcitol versus calcitriol on top of ACE-inhibitor therapy in the COL4A3 knockout mouse model for progressive renal fibrosis.

BACKGROUND: The COL4A3-/- mouse serves as animal model for progressive renal fibrosis. Using this animal model, the present study investigates the nephroprotective effects of Paricalcitol versus Calcitriol alone and on top of ACE-inhibitor therapy. METHODS: Eighty six mice were divided into six groups: (PC) with Paricalcitol 0.1 mcg/kg, (CA) Calcitriol 0.03 mcg/kg (dose equipotent), (PLAC) vehicle 0.1 mL i.p. five times per week, (ACE + PC) Paricalcitol plus Ramipril, (ACE + CA) Calcitriol plus Ramipril and (ACE + PLAC) vehicle plus Ramipril 10 mg/kg/day p.o. ACE therapy started pre-emptively in Week 4, PC/CA therapy was initiated in 6-week-old animals with ongoing renal fibrosis and lasted for 8 weeks. Four to six animals were sacrificed after 9.5 weeks and kidneys were further investigated using histological, immunohistological and Western-blot techniques. Survival until end-stage renal failure was determined in the remaining animals. RESULTS: PC, but not CA, prolonged lifespan until renal failure by 13% compared with untreated controls (P = 0.069). ACE-inhibition prolonged lifespan by >50%. Added on top of ACE inhibition, ACE + PC (but not ACE + CA) even further prolonged lifespan by additional 18.0% (P < 0.01 versus ACE + PLAC) and improved renal function (blood urea nitrogen; P < 0.05 versus ACE + CA). Accumulation of extracellular matrix and renal scarring was decreased in PC and ACE + PC-treated mice. CONCLUSIONS: The present study demonstrated a substantial nephroprotective and antifibrotic effect of the vitamin D-receptor activator Paricalcitol on top of early ACE inhibition in the COL4A3-/- model of progressive kidney fibrosis. The synergistic effect of Paricalcitol on top of RAAS-blockade might as well be valuable in other chronic kidney diseases.

Cell-matrix interactions in muscle disease.

The extracellular matrix (ECM) provides a solid scaffold and signals to cells through ECM receptors. The cell-matrix interactions are crucial for normal biological processes and when disrupted they may lead to pathological processes. In particular, the biological importance of ECM-cell membrane-cytoskeleton interactions in skeletal muscle is accentuated by the number of inherited muscle diseases caused by mutations in proteins conferring these interactions. In this review we introduce laminins, collagens, dystroglycan, integrins, dystrophin and sarcoglycans. Mutations in corresponding genes cause various forms of muscular dystrophy. The muscle disorders are presented as well as advances toward the development of treatment.

Mannose receptor 2 attenuates renal fibrosis.

Mannose receptor 2 (Mrc2) expresses an extracellular fibronectin type II domain that binds to and internalizes collagen, suggesting that it may play a role in modulating renal fibrosis. Here, we found that Mrc2 levels were very low in normal kidneys but subsets of interstitial myofibroblasts and macrophages upregulated Mrc2 after unilateral ureteral obstruction (UUO). Renal fibrosis and renal parenchymal damage were significantly worse in Mrc2-deficient mice. Similarly, Mrc2-deficient Col4α3(-/-) mice with hereditary nephritis had significantly higher levels of total kidney collagen, serum BUN, and urinary protein than Mrc2-sufficient Col4α3(-/-) mice. The more severe phenotype seemed to be the result of reduced collagen turnover, because procollagen III (α1) mRNA levels and fractional collagen synthesis in the wild-type and Mrc2-deficient kidneys were similar after UUO. Although Mrc2 associates with the urokinase receptor, differences in renal urokinase activity did not account for the increased fibrosis in the Mrc2-deficient mice. Treating wild-type mice with a cathepsin inhibitor, which blocks proteases implicated in Mrc2-mediated collagen degradation, worsened UUO-induced renal fibrosis. Cathepsin mRNA profiles were similar in Mrc2-positive fibroblasts and macrophages, and Mrc2 genotype did not alter relative cathepsin mRNA levels. Taken together, these data establish an important fibrosis-attenuating role for Mrc2-expressing renal interstitial cells and suggest the involvement of a lysosomal collagen turnover pathway.

A cell-molecular approach predicts vertebrate evolution.

In contrast to the conventional use of genes to determine the evolution of phenotypes, we have functionally integrated epithelial-mesenchymal interactions that have facilitated lung phylogeny and ontogeny in response to major geologic epochs. As such, this model reveals the underlying principles of lung physiology based on the evolutionary interactions between internal and external selection pressures, providing a novel understanding of lung biology. As a result, it predicts how cell-molecular changes in this process can cause disease and offers counterintuitive insights to diagnosis and treatment based on evolutionary principles.

Mechanotransduction through fibronectin-integrin focal adhesion in microvascular smooth muscle cells: is calcium essential?

It is believed that increased transmural pressure exerts force on vascular smooth muscle cells (VSMCs) and triggers Ca(2+) signaling as an initiating event responsible for the arteriolar myogenic response. However, the mechanisms linking the pressure increase to Ca(2+) signaling are unclear. We have shown previously using atomic force microscopy (AFM) that mechanical force induces a VSMC contractile response when applied to single fibronectin (FN; Sun Z, Martinez-Lemus LA, Hill MA, Meininger GA. Am J Physiol Cell Physiol 295; C268-C278, 2008) focal adhesion sites. This current study seeks to determine whether application of force to single focal adhesions can cause a change in VSMC Ca(2+). Experiments were performed in low passage (p3∼10) as well as in freshly isolated skeletal muscle arteriole VSMCs. AFM-attached microbeads (5 µm) were coated with FN or collagen type I (CN-I) or type IV (CN-IV) and placed on a VSMC for 20 min, resulting in formation of a focal adhesion between the cell and the microbead. In low passage VSMCs, mechanically pulling on the FN-coated beads (800∼3000 pN) did not induce a Ca(2+) increase but did cause a contractile response. In freshly isolated VSMCs, application of an FN or CN-I-coated bead onto the cell surface induced global Ca(2+) increases. However, these Ca(2+) increases were not correlated with the application of AFM pulling force to the bead or with the VSMC contractile responses to FN-coupled pulling. Chelating cytosolic Ca(2+) using BAPTA loading had no negative effect on the focal adhesion-related contractile response in both freshly isolated and low passage VSMCs, while the Rho-kinase inhibitor Y27632 abolished the micromyogenic response in both cases. These observations suggest that, in freshly isolated and cultured VSMCs, application of mechanical force to a focal adhesion does not invoke an acute global Ca(2+) increase. On the other hand, our data support a role for Rho-linked signaling mechanism involved in mechanotransduction leading to focal contraction that is independent of the need for a global increase in VSMC Ca(2+).

Matrix reloaded to circulation hits the tumor target.

Proteolytic fragments of various components of the extracellular matrix exhibit antiangiogenic activity via interaction with endothelial cell surface integrins. Kalluri and coworkers (this issue of Cancer Cell) use gene-targeted mice to show a physiological role for a carboxy-terminal fragment of collagen IV in the regulation of tumor angiogenesis.