RESUMO
The morphological and functional differences between the retinal and choroidal vascular bed raise the question of whether the smallest functional unit, the microvascular endothelial cell (MVEC), also differs in its basal characteristics. Here, we examined bovine retinal and choroidal MVECs (rMVECs, cMVECs) for the presence and regulation of angiogenic mediators and their receptors, and cytokines at the mRNA level using quantitative RT-PCR and differential display. Vascular endothelial growth factor (VEGF) mRNA was expressed in both rMVECs and cMVECs. The basal and hypoxia-increased VEGF mRNA levels were significantly higher in cMVECs, which may indicate a higher capacity for autocrine stimulation in these cells. The mRNA for two VEGF receptors, Flt-1 and Flk-1, was present in rMVECs and cMVECs. Interestingly, rMVECs expressed higher Flt-1 but lower Flk-1 mRNA levels compared to cMVECs. Examining the angiopoietin (Ang)/Tie-2 system, we only detected Ang-1 mRNA at very low levels. While Ang-2 mRNA levels were high in both rMVECs and cMVECs, rMVECs expressed 2-3 times the basal and hypoxia-upregulated Ang-2 mRNA levels than did cMVECs. No difference was found in basal Tie-2 mRNA levels. rMVECs are the more potent producers of macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF), whereas cMVECs expressed higher RANTES mRNA levels. In our second approach - screening rMVECs and cMVECs for differentially expressed genes - we found liprin-beta1, calnexin, and sushi-repeat-containing protein, x chromosome (SRPX) mRNA in both MVEC types at varying levels. In summary, MVECs from the retinal and choroidal vascular beds showed quantitative differences in angiogenic regulator expression and in their capability to produce cytokines.
Assuntos
Corioide/irrigação sanguínea , Endotélio Vascular/metabolismo , Vasos Retinianos/metabolismo , Animais , Bovinos , Hipóxia Celular/fisiologia , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Endotélio Vascular/citologia , Expressão Gênica , Microcirculação/metabolismo , RNA Mensageiro/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Phenotypically and functionally different types of microvascular endothelial cells (MVECs) derived from the developing corpus luteum were isolated and characterized by our group. We investigated whether these cytokeratin-positive (CK+) and cytokeratin-negative (CK-) MVECs differed in the expression of angiogenic factors and their regulation under hypoxia. Using quantitative RT-PCR, VEGF and its receptors, Flk-1 and Flt-1, were detected in CK- MVECs. The mRNA expression of Flk-1 mRNA was 100 times as high as that of Flt-1 mRNA. CK+ MVECs expressed VEGF and Flt-1 mRNA, but were devoid of Flk-1 transcripts. No Ang-1 mRNA was demonstrated in either cell type, and Ang-2 mRNA was found only in CK- MVECs. Tie-2 mRNA was detected in both MVEC types, but levels were 150 times as high in CK- MVECs as in CK+ MVECs. mRNA of hypoxia-inducible factors Hif-1alpha and Hif-1beta was expressed in both MVEC types. After hypoxia, neither VEGF, nor Flk-1, nor Tie-2 mRNA expression was altered in either MVEC type. Flt-1 expression and Ang-2 mRNA expression were significantly increased at about 2.5-fold (P < 0.05) in CK- MVECs, but not in CK+ MVECs. Our study demonstrates the varying expression and regulation of angiogenesis-related factors and receptors in phenotypically different MVEC types.