Vascular smooth muscle cell differentiation in human cerebral vascular malformations.

OBJECTIVE: The pathogenesis of central nervous system vascular malformations likely involves the abnormal assembly, differentiation of vascular smooth muscle cells (VSMC), or both in association with dysmorphic vessel wall. We hypothesize that intracranial arteriovenous malformations (AVMs) and cerebral cavernous malformations (CCMs) exhibit distinct patterns of expression of molecular markers of differentiation and maturity of VSMCs. We further speculate that the unique VSMC phenotype in the different lesions is not necessarily maintained in cell culture. METHODS: Paraffin-embedded sections of five AVMs, CCMs, and control brain tissues were stained immunohistochemically with antibodies to alpha-smooth muscle actin (alpha-SMA), myosin heavy chain, and smoothelin, a novel marker for contractile VSMC phenotype. Large (> or =100 microm) and small (<100 microm) vessels were counted and assessed for immunoexpression of each protein, then categorized according to expression of one or more of these markers. Cultured nonendothelial cells isolated from four other excised AVM and CCM lesions were assessed for immunoexpression of the same antibodies. RESULTS: Alpha-SMA was universally expressed in all vessels in AVMs and in control brains. It was expressed in the subendothelial layer of 97% of large caverns and 85% of small caverns and in scattered intercavernous connective tissue fibrocytes in CCMs. Myosin heavy chain was expressed in the majority of brain and AVM vessels, except for normal veins, and in the subendothelial layer of more than half of the caverns in CCMs. Smoothelin expression was less prevalent in large vessels in AVMs than in control brains and was not found in any caverns in CCMs (large vessels in control brains, 40.9%; AVMs, 21.9%; CCMs, 0%; P < 0.0001). Cultured AVM and CCM nonendothelial cells expressed alpha-SMA, but myosin heavy chain was expressed weakly in cells from only one CCM. Smoothelin was negative in all cells. CONCLUSION: We describe vessels with various stages of VSMC differentiation in AVMs and CCMs. The subendothelial layer of CCMs commonly expresses alpha-SMA and less commonly expresses myosin heavy chain. Expression of smoothelin was less prevalent in large AVM vessels than in normal brain, which may reflect the loss of contractile property associated with hemodynamic stress. It is difficult to evaluate VSMC differentiation in culture because of phenotypic change.

Expression of endothelial cell angiogenesis receptors in human cerebrovascular malformations.

OBJECTIVE: To further understand the role of angiogenic growth factors in the development of cerebral cavernous malformations (CCMs) and arteriovenous malformations (AVMs), we investigated endothelial cell (EC) expression of receptors for vascular endothelial growth factor (VEGF) and angiopoietin systems in patients with surgically resected lesions. METHODS: Paraffin-embedded sections of five AVMs, CCMs, and normal control brain tissue samples were stained immunohistochemically with antibodies to von Willebrand factor and CD31 (to characterize ECs) and angiogenesis growth factor receptors Flt-1 (VEGF-R1), Flk-1 (VEGF-R2), Tie-1, and Tie-2. We counted large and small vessels in each specimen, assessed each specimen's immunoexpression of each antigen, and analyzed differences between CCMs, AVMs, and the normal control brain tissue samples. RESULTS: The ECs of CCMs, AVMs, and normal control brain tissue samples expressed the von Willebrand factor uniformly, but the ECs of CCMs were largely negative for CD31 (P < 0.05). Flk-1, Flt-1, and Tie-2 were not expressed in the control brain tissue samples. The proportion of immunopositive vessels to VEGF receptors Flk-1 and Flt-1 was significantly greater in AVMs and CCMs than in the control brain tissue samples (P < 0.05). Tie-2 in AVMs and CCMs was expressed in a higher percentage of immunopositive vessels than in the control brain tissue samples, but the difference was not statistically significant. Tie-1 was expressed in rare vessels of all lesion types and control brain tissue samples. CONCLUSION: ECs of CCMs do not seem to express CD31 to the same extent that AVMs and normal brain tissue do. AVMs and CCMs show greater expression of VEGF receptors, but not of angiopoietin receptors, than normal brain tissue does.

Endothelial cell culture from human cerebral cavernous malformations.

BACKGROUND AND PURPOSE: The cerebral cavernous malformation (CCM) is a common and frequently unrecognized cause of stroke and epilepsy. It consists of blood-filled caverns lined by endothelial cells (EC) and devoid of mature vessel wall structure. Cultured EC obtained from CCM may express phenotypic and genotypic alterations contributing to CCM pathogenesis. We report the first successful isolation and growth in vitro of primary EC lines from human CCM lesions. METHODS: We developed a procedure for the isolation and growth of EC from human CCM, confirmed their EC origin by a panel of molecular markers, and determined by immunocytochemistry the basic expression patterns of 6 transmembrane receptor protein kinases comparing brain, skin, and CCM primary EC lines grown identically. RESULTS: Several CCM EC lines were established from 2 patients after we treated the excised specimens with 0.3% trypsin/1% EDTA, selective cloning, and growth in MCDB107 containing 0.3 g/L heparin, 0.15 g/L endothelial cell growth supplement, and 15% FBS. The CCM EC showed contact inhibition and a rounded cobblestone appearance. The cells expressed CD31, CD105, von Willebrand factor, and binding sites for Ulex europaeus agglutinin, type 1 and acetylated LDL. They showed low levels of Flt-1, Flk-1, transforming growth factor (TGF)-beta RI, and TGF-beta RII expression but stained strongly with antibodies against Tie-1 and Tie-2. CONCLUSIONS: Cultured CCM EC retained their endothelial phenotype. Brain, skin, and CCM EC lines did not significantly differ in their staining patterns with antibodies against Flt-1, Flk-1, TGF-beta RI, TGF-beta RII, Tie-1, and Tie-2. These cell lines will assist in defining molecular phenotype and genotype alterations in association with CCM.