Behavior of endothelial cells on Matrigel and development of a method for a rapid and reproducible in vitro angiogenesis assay.

During the process of angiogenesis, the normally quiescent endothelial cells that line the vasculature are induced to proliferate, migrate and align to form new blood vessels by angiogenic stimuli. Assays for angiogenic factors mostly involve in vivo approaches. The two most commonly used in vivo assays-the chick chorioallantoic membrane (CAM) assay and the rabbit corneal assay are tedious to perform and are technically demanding. Several in vitro assays have also been developed, based on the ability of endothelial cells to form tubes in 3-D matrices. Here, we describe the modification of a microcarrier bead-based assay. This assay combines cells grown on Cytodex-3 microcarrier beads with Matrigel to provide an easy, rapid, and reliable method for evaluating and measuring angiogenic activity. We also describe the differential behavior of normal and transformed endothelial cells cultured in Matrigel.

Human angiogenin is a neuroprotective factor and amyotrophic lateral sclerosis associated angiogenin variants affect neurite extension/pathfinding and survival of motor neurons.

Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disorder affecting upper and lower motor neurons (MNs). The molecular mechanisms underlying ALS are poorly understood. Mutations in SOD1 is one of the known causes of ALS but occur only in a very small number of cases of ALS. Interestingly, mutations in human angiogenin (hANG), a member of the ribonuclease A (RNase A) superfamily known to be involved in neovascularization, have been recently reported in patients with ALS, but the effects of these mutations on MN differentiation and survival has not been investigated. We have used the well-characterized pluripotent P19 embryonal carcinoma (EC) cell culture model of neuro-ectodermal differentiation to study the effects of hANG-ALS variants on MN differentiation and survival. Here we report that P19 EC cells induced to differentiate in the presence of hANG and hANG-ALS-associated variants internalize the wild-type and variant proteins. The P19 EC cells differentiate to form neurons but the ability of the neurites to extend and make contacts with neighbouring neurites is compromised when treated with the hANG-ALS variants. In addition, hANG-ALS variants also have a cytotoxic effect on MNs leading to their degeneration. hANG was able to protect neurons from hypoxia-induced cell death, but the variants of hANG implicated in ALS lacked the neuroprotective activity. Our findings show that ANG plays an important role in neurite extension/pathfinding and survival providing a causal link between mutations in hANG and ALS.

Biological and structural features of murine angiogenin-4, an angiogenic protein.

Murine angiogenin-4 (mAng-4) is a member of the pancreatic ribonuclease superfamily that is expressed in some endodermally derived organs. We now show that mAng-4 is angiogenic using a thoracic aorta assay never before applied to the angiogenins. mAng-4, human angiogenin (hAng), and murine angiogenin-1 (mAng-1) stimulate the proliferation of IGR1 melanoma cells but do not stimulate the proliferation or migration of bovine corneal endothelial cells or primary mouse embryonic fibroblasts. In addition, we report the 3-D structure of mAng-4 at 2.02-A resolution. The structure shows that the residues forming the putative B1, P1, and B2 RNA-binding subsites occupy positions similar to their hAng counterparts. The B1 subsite is obstructed by Glu115 and Ile118. The obstruction is stabilized by a novel salt bridge between the C-terminal carboxyl group and the side chain of Arg99. Through mutational studies, we identify residues critical to the angiogenic function of mAng-4. The effect of H12A and H112A mutations in the catalytic site indicates that ribonucleolytic activity is essential to angiogenesis. The consequences of a nearby E115A mutation are consistent with a significant role for Glu115 in the attenuation of enzymatic activity but also suggest that sufficient suppression of catalysis is necessary for angiogenesis. The effect of an R32A mutation in the putative nuclear localization sequence indicates that this residue is crucial for angiogenesis. In the putative cell-binding segment, the replacement of Lys59 with Asn (its counterpart at position 61 of hAng) does not abrogate enzymatic activity but abolishes angiogenic activity, the reason for which is unclear.

Characterization of human angiogenin variants implicated in amyotrophic lateral sclerosis.

Human angiogenin (ANG), the first member of the angiogenin family (from the pancreatic ribonuclease A superfamily) to be identified, is an angiogenic factor that induces neovascularization. It has received much attention due to its involvement in the growth of tumors and its elevated expression level in pancreatic and several other cancers. Recently the biological role of ANG has been shown to extend to the nervous system. Mutations in ANG have been linked with familial as well as sporadic forms of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by selective destruction of motor neurons. Furthermore, mouse angiogenin-1 has been shown to be expressed in the developing nervous system and during the neuronal differentiation of pluripotent stem cells. We have now characterized the seven variants of ANG reported in ALS patients with respect to the known biochemical properties of ANG and further studied the biological properties of three of these variants. Our results show that the ribonucleolytic activity of six of the seven ANG-ALS implicated variants is significantly reduced or lost and some variants also show altered thermal stability. We report a significant reduction in the cell proliferative and angiogenic activities of the three variants that we chose to investigate further. Our studies on the biochemical and structural features of these ANG variants now form the basis for further investigations to determine their role(s) in ALS.