Ken-ichiro Tashiro

Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro

Endothelial cells, both microvascular as well as large vessel, undergo differentiation slowly in culture under most conditions. When endothelial cells are cultured on Matrigel, a solid gel of basement membrane proteins, they rapidly align and form hollow tube-like structures. We show here that tube formation is a multi-step process induced by laminin. An RGD-containing sequence in the A chain of laminin through an integrin receptor on the endothelial cell induces their attachment to the protein while a YIGSR site in the B1 chain induces cell-cell interactions and the resulting tube formation. We also show that the laminin-derived synthetic peptide YIGSR contains sufficient information to induce single endothelial cells to form ring-like structures surrounding a hollow lumen, the basic putative unit in the formation of capillaries.

Laminin A chain synthetic peptide which supports neurite outgrowth

Neurons from peripheral and central nervous tissue as well as from established cell lines respond to low concentrations of laminin with rapid extension of axon-like processes. Two sites on laminin have been identified which stimulate neurite outgrowth, the major site residing at the end of the long arm of laminin. Recently laminin has been cloned and sequenced allowing for synthetic peptides to be prepared and tested for biological activity. We report here that antisera against synthetic peptides corresponding to A and to B1 chain sequences at the end of the long arm can partially inhibit laminin-mediated neurite outgrowth. Further, we show that a 19 amino acid synthetic peptide (CSRARKQAASIKVAVSADR) from the long arm of the laminin A chain is capable of stimulating neuronal-like process formation to almost the same extent as laminin and competes with laminin for stimulation of neurite outgrowth.

The relationship between the aggregational state of the amyloid-β peptides and free radical generation by the peptides

In the present study, we investigated whether or not the amyloid‐β protein (Aβ) peptide itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of Aβ and Aβ‐induced cytotoxicity. The present results demonstrated a four‐line spectrum in the presence of both Aβ40 and Aβ42 with Ntert‐butyl‐α‐phenylnitrone (PBN), but not in the presence of PBN alone in phosphate‐buffered saline (PBS). The fact that the four‐line spectrum obtained for the Aβ/PBN in PBS was completely abolished in the presence of the iron‐chelating agent Desferal demonstrated the observed four‐line spectrum to be iron‐dependent. The present study also revealed that either Aβ40 or Aβ42 with PBN in phosphate buffer (PB) did not produce any definite four‐line spectrum. Both a thioflavine‐T (Th‐T) fluorometric assay and circular dichroism (CD) spectroscopy showed the amyloid fibril formation of Aβ in PBS to be much higher than that of Aβ in PB. Moreover, Aβ‐induced cytotoxicity assays showed Aβ incubated in PBS to be more cytotoxic than that incubated in PB. These results thus suggest that Aβ‐associated free radical generation is strongly influenced by the aggregational state of the peptides.

Laminin in neuronal development

Laminin is the major glycoprotein in basement membranes, the thin extracellular matrix that underlies epithelial and endothelial cells and surrounds nerves, muscle, and fat cells.’-3 It was first described in 1979 and later found to have many important biological activities: Laminin promotes cell adhesion, growth, migration, differentiation, collagenase IV production, and phagocytosis; and it enhances the metastatic phenotype of malignant cells. It is also a potent promoter of neurite outgrowth. Primary and established neural cells from both the peripheral and central nervous systems respond to laminin with increased adhesion and by extending long neuritelike processes (reviewed in Ref. 5) .

A laminin-pepsin fragment with cell attachment and neurite outgrowth activity at distinct sites

Laminin is a large basement membrane glycoprotein which influences the behavior and morphology of a variety of cells. We have found that laminin and a pepsin fragment of laminin (P-lam) contain distinct sites for HT-1080 human fibrosarcoma cell attachment and for neurite outgrowth activity of PC12 and NG108-15 cell lines. Reduction and alkylation of laminin and P-lam fragment disulfide bonds, in the absence of denaturing agents, markedly reduced the cell attachment activity without reducing the neurite outgrowth response. The P-lam fragment (approximately 375 kDa) was found to contain part of the cross region of laminin and a portion of the long arm, on the basis of recognition by antisera against laminin synthetic peptides and fusion proteins. Modification of arginine residues by cyclohexanedione also had no effect on neurite outgrowth but reduced HT-1080 cell adhesion. Modification of lysine residues by succinic and citraconic anhydride, however, abolished laminin neurite outgrowth but not cell attachment activity. Neurite outgrowth activity was recovered by reversing the lysine modification. These data support the existence on laminin of separate sites for cell attachment and for neurite outgrowth.

Inhibition of Aβ fibril formation and Aβ-induced cytotoxicity by senile plaque-associated proteins

Aβ neurotoxicity is generally believed to require Aβ fibril formation. The prevention of Aβ fibril formation thus seems to be a promising strategy for the treatment of AD. Recent studies have shown senile plaque-associated proteins such as laminin to have an inhibitory effect on both Aβ40 and Aβ42 fibril formation in vitro. In the present study, we thus investigated whether or not midkine (MK) and α2-macroglobulin (α2M), both of which are also senile plaque-associated proteins like laminin, affect Aβ fibril formation and Aβ-induced cytotoxicity. The present study demonstrated that both MK and α2M inhibit both Aβ fibril formation and Aβ-induced cytotoxicity in PC12 cells. The confirmation of the present results based on in vivo experiments is called for in future studies to clarify whether or not senile plaque-associated proteins such as MK and α2M can be a model for therapeutic agents in the treatment of AD.

Laminin inhibits both Aβ40 and Aβ42 fibril formation but does not affect Aβ40 or Aβ42-induced cytotoxicity in PC12 cells

Laminin has recently been reported to inhibit both Aβ40 and Aβ42 fibril formation in vitro. Laminin was thus suggested to be an effective therapeutic agent for Alzheimers disease. However, some recent reports have shown that Aβ fibril formation may not necessarily be linked to the development of Aβ neutotoxicity. In the present study, we thus investigated whether or not laminin affects Aβ40 and Aβ42-induced neurotoxicity. The findings of the present study by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye reduction test showed laminin not to have an inhibitory effect on Aβ40 or Aβ42-induced cytotoxicity in PC12 cells while Aβ fibril formation was inhibited under the conditions used in the present study. The findings of the present study therefore do not support the hypothesis that Aβ fibril formation is absolutely required for the development of Aβ cytotoxicity.

Laminin inhibits Aβ42 fibril formation in vitro

Abstract In the present study, we investigated whether or not laminin inhibits A β 42 fibril formation in the same manner as A β 40. Both a thioflavine-T fluorometric assay and electron microscopy by negative staining demonstrated laminin to have a concentration-dependent inhibitory effect on A β 42 fibril formation. The amyloid fibril formation was inhibited approximately by 70% due to the presence of 1.0 mg/ml laminin co-incubated with 1.0 mg/ml A β 42 peptide (molar ratio; A β 42 peptide:laminin=200:1). These results thus suggested that laminin or its derivatives may be effective as therapeutic agents to either prevent or slow down the progression of amyloidogenesis in Alzheimers disease.

Identification of a heparin binding site and the biological activities of the laminin α1 chain carboxy-terminal globular domain

The carboxy‐terminal globular domain (G‐domain) of the laminin α1 chain has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. In this study, we defined the potential sequences originating from the G‐domain of laminin α1 chain which possess these functional activities. A series of peptides were synthesized from the G‐domain, termed LG peptides (LG‐1 to LG‐6) and were tested for their various biological activities. In the direct [3H]heparin binding assays, LG‐6 (residues 2,335–2,348: KDFLSIELVRGRVK) mediated high levels of [3H]heparin binding, and this peptide also directly promoted cell adhesion and spreading, including B16F10, M2, HT1080, and PC12 cells. The peptide LG‐6 also promoted the neurite outgrowth of PC12 cells, mouse granule cells, and chick telencephalic cells. An anti‐peptide LG‐6 antibody inhibited laminin‐1 and peptide LG‐6–mediated cell adhesion and neurite outgrowth. Furthermore, an anti‐integrin α2 antibody also inhibited the cell adhesion activity. These results suggest that peptide LG‐6 plays a functional role as a heparin binding site in the G‐domain of the laminin α1 chain, and this sequence was thus concluded to play a crucial role in regulating cell adhesion and spreading and neurite outgrowth which is related to integrin α2. J. Cell. Physiol. 179:18–28, 1999.

Laminin inhibits Aβ40 fibril formation promoted by apolipoprotein E4 in vitro

Abstract The aggregation of soluble Aβ into insoluble amyloid fibrils is believed to be an important step in the pathogenesis of Alzheimers disease (AD) and the prevention of this process therefore seems to be a promising strategy for the treatment of AD. Both apolipoprotein E(apoE) and laminin are known to play important roles in the regeneration of the central nervous system and both are known to accumulate in the senile plaques of the AD brains. In the present study, we therefore investigated whether or not laminin has any effect on Aβ40 fibril formation promoted by apoE4 in vitro. A thioflavine-T fluorometric assay and electron microscopic observations using negative staining together demonstrated that laminin inhibits Aβ40 fibril formation in vitro while it also inhibits Aβ40 fibril formation promoted by apoE4. These results suggested that either laminin or its derivatives may thus be effective as therapeutic agents for AD.