Yamato Kikkawa

Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases

Kidney disease affects over 20 million people in the United States alone. Although the causes of renal failure are diverse, the glomerular filtration barrier is often the target of injury. Dysregulation of VEGF expression within the glomerulus has been demonstrated in a wide range of primary and acquired renal diseases, although the significance of these changes is unknown. In the glomerulus, VEGF-A is highly expressed in podocytes that make up a major portion of the barrier between the blood and urinary spaces. In this paper, we show that glomerular-selective deletion or overexpression of VEGF-A leads to glomerular disease in mice. Podocyte-specific heterozygosity for VEGF-A resulted in renal disease by 2.5 weeks of age, characterized by proteinuria and endotheliosis, the renal lesion seen in preeclampsia. Homozygous deletion of VEGF-A in glomeruli resulted in perinatal lethality. Mutant kidneys failed to develop a filtration barrier due to defects in endothelial cell migration, differentiation, and survival. In contrast, podocyte-specific overexpression of the VEGF-164 isoform led to a striking collapsing glomerulopathy, the lesion seen in HIV-associated nephropathy. Our data demonstrate that tight regulation of VEGF-A signaling is critical for establishment and maintenance of the glomerular filtration barrier and strongly supports a pivotal role for VEGF-A in renal disease.

Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin α5 in the glomerular basement membrane

In developing glomeruli, laminin α5 replaces laminin α1 in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. To investigate domain-specific functions of laminin α5 during glomerulogenesis, we produced transgenic mice that express a chimeric laminin composed of laminin α5 domains VI through I fused to the human laminin α1 globular (G) domain, designated Mr51. Transgene-derived protein accumulated in many basement membranes, including the developing GBM. When bred onto the Lama5 −/− background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 −/− glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells. However, here the phenotype could be attributed to the lack of mesangial cell adhesion to the GBM, suggesting that the G domain of laminin α5 is essential for this adhesion. Analysis of an additional chimeric transgene allowed us to narrow the region of the α5 G domain essential for mesangial cell adhesion to α5LG3-5. Finally, in vitro studies showed that integrin α3β1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin α5. Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin α5 in the GBM.

Blood vessels of human islets of Langerhans are surrounded by a double basement membrane

Aims/hypothesisBased on mouse study findings, pancreatic islet cells are supposed to lack basement membrane (BM) and interact directly with vascular endothelial BM. Until now, the BM composition of human islets has remained elusive.MethodsImmunohistochemistry with specific monoclonal and polyclonal antibodies as well as electron microscopy were used to study BM organisation and composition in human adult islets. Isolated islet cells and function-blocking monoclonal antibodies and recombinant soluble Lutheran peptide were further used to study islet cell adhesion to laminin (Lm)-511. Short-term cultures of islets were used to study Lutheran and integrin distribution.ResultsImmunohistochemistry revealed a unique organisation for human Lm-511/521 as a peri-islet BM, which co-invaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained Lm-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the Lm α5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR, whereas α3 and β1 integrins were more diffusely distributed. High Lutheran content was also found on endocrine cell membranes in short-term culture of human islets. The adhesion of dispersed beta cells to Lm-511 was inhibited equally effectively by antibodies to integrin and α3 and β1 subunits, and by soluble Lutheran peptide.Conclusions/interpretationThe present results disclose a hitherto unrecognised BM organisation and adhesion mechanisms in human pancreatic islets as distinct from mouse islets.

Review: Lutheran/B-CAM: A Laminin Receptor on Red Blood Cells and in Various Tissues

The Lutheran blood group glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is a transmembrane receptor with five immunoglobulin-like domains in its extracellular region; it is therefore classified as a member of the immunoglobulin (Ig) gene family. Lu/B-CAM is observed not only on red blood cells, but also on a subset of muscle and epithelial cells in various tissues. Recently, several groups have reported that Lu/B-CAM is a novel receptor for laminin α5. The laminin α5 chain is a component of the laminin-511 (α5β1γ1), -521 (α5β2γ1), and -523 (α5β2γ3) heterotrimers and is expressed throughout the mammalian body. We also have shown that Lu/B-CAM is co-localized with laminin α5 in various tissues. Although the biological role of Lu/B-CAM remains unclear, the specific binding of Lu/B-CAM to laminin α5 suggests that it plays an important role in developmental and physiological processes. It also is necessary to investigate further the interaction between Lu/B-CAM and laminin α5 in pathological processes, including sickle cell disease and cancer.

The LG1-3 tandem of laminin alpha 5 harbors the binding sites of lutheran/basal cell adhesion molecule and alpha 3 beta 1/alpha 6 beta 1 integrins

The laminin-type globular (LG) domains of laminin α chains have been implicated in various cellular interactions that are mediated through receptors such as integrins, α-dystroglycan, syndecans, and the Lutheran blood group glycoprotein (Lu). Lu, an Ig superfamily transmembrane receptor specific for laminin α5, is also known as basal cell adhesion molecule (B-CAM). Although Lu/B-CAM binds to the LG domain of laminin α5, the binding site has not been precisely defined. To better delineate this binding site, we produced a series of recombinant laminin trimers containing modified α chains, such that all or part of α5LG was replaced with analogous segments of human laminin α1LG. In solid phase binding assays using a soluble Lu (Lu-Fc) composed of the Lu extracellular domain and human IgG1 Fc, we found that Lu bound to Mr5G3, a recombinant laminin containing α5 domains LN through LG3 fused to human laminin α1LG4–5. However, Lu/B-CAM did not bind other recombinant laminins containing α5LG3 unless α5LG1–2 was also present. A recombinant α5LG1-3 tandem lacking the laminin coiled coil (LCC) domain did not reproduce the activity of Lu/B-CAM binding. Therefore, proper structure of the α5LG1-3 tandem with the LCC domain was essential for the binding of Lu/B-CAM to laminin α5. Our results also suggest that the binding site for Lu/B-CAM on laminin α5 may overlap with that of integrins α3β1 and α6β1.

Laminin β2 Gene Missense Mutation Produces Endoplasmic Reticulum Stress in Podocytes

Mutations in the laminin β2 gene (LAMB2) cause Pierson syndrome, a severe congenital nephrotic syndrome with ocular and neurologic defects. LAMB2 is a component of the laminin-521 (α5β2γ1) trimer, an important constituent of the glomerular basement membrane (GBM). The C321R-LAMB2 missense mutation leads to congenital nephrotic syndrome but only mild extrarenal symptoms; the mechanisms underlying the development of proteinuria with this mutation are unclear. We generated three transgenic mouse lines, in which rat C321R-LAMB2 replaced mouse LAMB2 in the GBM. During the first postnatal month, expression of C321R-LAMB2 attenuated the severe proteinuria exhibited by Lamb2(-/-) mice in a dose-dependent fashion; proteinuria eventually increased, however, leading to renal failure. The C321R mutation caused defective secretion of laminin-521 from podocytes to the GBM accompanied by podocyte endoplasmic reticulum (ER) stress, likely resulting from protein misfolding. Moreover, ER stress preceded the onset of significant proteinuria and was manifested by induction of the ER-initiated apoptotic signal C/EBP homologous protein (CHOP), ER distention, and podocyte injury. Treatment of cells expressing C321R-LAMB2 with the chemical chaperone taurodeoxycholic acid (TUDCA), which can facilitate protein folding and trafficking, greatly increased the secretion of the mutant LAMB2. Taken together, these results suggest that the mild variant of Pierson syndrome caused by the C321R-LAMB2 mutation may be a prototypical ER storage disease, which may benefit from treatment approaches that target the handling of misfolded proteins.

The LG1-3 tandem of laminin α5 harbors the binding sites of Lutheran/B-CAM and α3β1/α6β1 integrins

The laminin-type globular (LG) domains of laminin α chains have been implicated in various cellular interactions that are mediated through receptors such as integrins, α-dystroglycan, syndecans, and the Lutheran blood group glycoprotein (Lu). Lu, an Ig superfamily transmembrane receptor specific for laminin α5, is also known as basal cell adhesion molecule (B-CAM). Although Lu/B-CAM binds to the LG domain of laminin α5, the binding site has not been precisely defined. To better delineate this binding site, we produced a series of recombinant laminin trimers containing modified α chains, such that all or part of α5LG was replaced with analogous segments of human laminin α1LG. In solid phase binding assays using a soluble Lu (Lu-Fc) composed of the Lu extracellular domain and human IgG1 Fc, we found that Lu bound to Mr5G3, a recombinant laminin containing α5 domains LN through LG3 fused to human laminin α1LG4–5. However, Lu/B-CAM did not bind other recombinant laminins containing α5LG3 unless α5LG1–2 was also present. A recombinant α5LG1-3 tandem lacking the laminin coiled coil (LCC) domain did not reproduce the activity of Lu/B-CAM binding. Therefore, proper structure of the α5LG1-3 tandem with the LCC domain was essential for the binding of Lu/B-CAM to laminin α5. Our results also suggest that the binding site for Lu/B-CAM on laminin α5 may overlap with that of integrins α3β1 and α6β1.

Laminin-111-derived peptides and cancer

Laminin-111 is a large trimeric basement membrane glycoprotein with many active sites. In particular, four peptides active in tumor malignancy studies have been identified in laminin-111 using a systematic peptide screening method followed by various assays. Two of the peptides (IKVAV and AG73) are found on the α1 chain, one (YIGSR) of the β1 chain and one (C16) on the γ1 chain. The four peptides have distinct activities and receptors. Since three of the peptides (IKVAV, AG73 and C16) strongly promote tumor growth, this may explain the potent effects laminin-111 has on malignant cells. The peptide, YIGSR, decreases tumor growth and experimental metastasis via a 32/67 kD receptor while IKVAV increases tumor growth, angiogenesis and protease activity via integrin receptors. AG73 increases tumor growth and metastases via syndecan receptors. C16 increases tumor growth and angiogenesis via integrins. Identification of such sites on laminin-111 will have use in defining strategies to develop therapeutics for cancer.

Laminin isoforms in human embryonic stem cells: synthesis, receptor usage and growth support.

To reveal the functional intrinsic niche of human embryonic stem cells (hESC) we examined the production of basement membrane (BM) proteins and the presence of their receptors in feeder‐free cell culture conditions. In addition, we investigated binding of hESCs to purified human BM proteins and identified the receptors mediating these contacts. Also, we tested whether purified human laminin (Lm) isoforms have a role in hESC self‐renewal and growth in short‐term cultures. The results show that hESCs synthesize Lm α1 and Lm α5 chains together with Lm β1 and γ1 chains suggesting the production of Lms‐111 and ‐511 into the culture medium and deposits on cells. hESCs contain functionally important integrin (Int) subunits, Int β1, α3, α6, α5, β5 and αV, as well as the Lm α5 receptor, Lutheran (Lu) glycoprotein and its truncated form, basal cell adhesion molecule (B‐CAM). In cell adhesion experiments, Int β1 was crucial for adhesion to most of the purified human BM proteins. Lu/B‐CAM mediated adhesion to Lm‐511 together with Int α3β1, and was essential for the adhesion of hESCs to embryonic feeder cells. Adhesion to Lm‐411 was mediated by Int α6β1. Lm‐511 supported hESC growth in defined medium equally well as Matrigel. These results provide consequential information of the biological role of BM in hESCs, warranting further investigation of BM biology of human pluripotent stem cells.

Laminin α5 mediates ectopic adhesion of hepatocellular carcinoma through integrins and/or Lutheran/basal cell adhesion molecule

Laminins are a diverse group of alpha/beta/gamma heterotrimers formed from five alpha, three beta and three gamma chains; they are major components of all basal laminae (BLs). One laminin chain that has garnered particular interest due to its widespread expression pattern and importance during development is laminin alpha 5. Little is known, however, about the expression and function of laminins containing the alpha 5 chain in human hepatocellular carcinoma (HCC). Here, using a specific antibody, we examined the expression of laminin alpha 5 in normal liver and in HCCs. In normal liver, although laminin alpha 5 was observed in hepatic BLs underlying blood vessels and bile ducts, it was absent from the parenchyma, which may be the origin of HCC. On the other hand, laminin alpha 5 deposition was observed throughout all HCCs tested, regardless of tumor grade. In well-differentiated HCCs, it localized along the trabecules of the tumor. In poorly-differentiated HCCs, it was present in surrounding tumor nodules. In HCC cell lines, laminin alpha 5 heterotrimerized with beta and gamma chains and was secreted into the culture media. To attempt to understand the function of laminins containing alpha 5, the expression of its receptors in HCCs was also determined. In this regard, alpha 3 beta 1/alpha 6 beta 1 integrins and Lutheran/basal cell adhesion molecule (Lu/B-CAM) were expressed in HCC cells. In vitro studies showed that HCC cells readily attached to laminin containing the alpha 5 chain, more so than did primary hepatocytes. In addition to alpha 3 beta 1/alpha 6 beta 1 integrins and Lu/B-CAM, laminin alpha 5 was recognized by integrin alpha 1 beta 1, which also was expressed in HCC cells. These results suggest that laminins containing alpha 5 serve as functional substrates regulating progression of HCC.