Aberrant regulation of retinoic acid signaling genes in cerebral arterio venous malformation nidus and neighboring astrocytes

Abstract

Background: Cerebral arterio venous malformations (AVM) are a major causal factor for intracranial hemorrhage, which result in permanent disability or death. The molecular mechanisms of AVM are complex, and their pathogenesis remains an enigma. Current research on cerebral AVM is focused on characterizing the molecular features of AVM nidus to elucidate the aberrant signaling pathways. The initial stimuli that lead to the development of AVM nidus structures between a dilated artery and a vein are however not known.

Methods: In order to understand the molecular basis of development of cerebral AVM, we used in-depth RNA sequencing with the total RNA isolated from cerebral AVM nidus. Immunoblot and qRT-PCR assays were used to study the differential gene expression in AVM nidus, and immunofluorescence staining was used to study the expression pattern of aberrant proteins in AVM nidus and control tissues. Immunohistochemistry was used to study the expression pattern of aberrant proteins in AVM nidus and control tissues.

Results: The transcriptome study has identified 38 differentially expressed genes in cerebral AVM nidus, of which 35 genes were upregulated and 3 genes were downregulated. A final modular analysis identified an upregulation of ALDH1A2, a key rate-limiting enzyme of retinoic acid signaling pathway. Further analysis revealed that CYR61, a regulator of angiogenesis, and the target gene for retinoic acid signaling is upregulated in AVM nidus. We observed that astrocytes associated with AVM nidus are abnormal with increased expression of GFAP and Vimentin. Triple immunofluorescence staining of the AVM nidus revealed that CYR61 was also overexpressed in the abnormal astrocytes associated with AVM tissue.

Conclusion: Using high-throughput RNA sequencing analysis and immunostaining, we report deregulated expression of retinoic acid signaling genes in AVM nidus and its associated astrocytes and speculate that this might trigger the abnormal angiogenesis and the development of cerebral AVM in humans.

Keywords: ALDH1A2; Astrocytes; Gene expression; Retinoic acid; Vascular system.

Fig. 1

Transcriptome analysis of cerebral AVM nidus and controls tissues. a Pie diagram showing gene distribution of differentially expressed genes in arterio venous malformation (AVM) nidus samples compared to control sample. b The scatter plot representation of differentially expressed genes in cerebral AVM nidus, which allows to identify the expression levels of genes in two distinct conditions. In the scatter plot, each dot represents a gene, and thus, genes that fall above the diagonal are overexpressed and genes that fall below the diagonal are underexpressed in AVM. c Top 100 differentially expressed genes in the cerebral AVM tissues compared to control brain tissues are represented as heat map. The asterisk marked genes represent the genes involved in blood vessel development. The color represents the logarithmic intensity of the expressed genes. Relatively high expression values are shown in red color. d The bar plot represents the Log2 fold change in the expression of commonly differentially expressed genes in the cerebral AVM samples compared to control samples. A total of 38 genes were differentially expressed commonly in all the three independent AVM samples analyzed compared to control samples, among which 35 genes were upregulated and three genes viz, NR5A1, SSTR1, and DUX4L4, were downregulated. e Validation of differentially expressed genes common in three independent samples of cerebral AVM nidus. The following genes were validated by qRT-PCR. COL3A1, CYR61, OSM, MAFB, COL1A1, CTSS, TLR2, and CXCR4, and their expression were normalized to control samples, and internal normalization was carried out using GAPDH. Many tested genes were significantly up regulated in AVM tissue. The LIF and MGP gene expression were not significantly increased in AVM compared to control tissue. The qRT-PCR was carried out with 10 AVM nidus and 10 control samples, and the GAPDH was used as endogenous control for normalization. Individual paired t test was performed to calculate the P values (*P < 0.05 and **P < 0.01)

Fig. 2

Analysis of aberrantly expressed retinoic acid-responsible genes in AVM nidus. a The heat map representing retinoic acid response genes from the RNA sequencing analysis that are differentially expressed in the cerebral AVM nidus. The genes that are directly or indirectly associated with vascular development are marked with an asterisk, and the list of marked genes are provided in the expanded version. b Validation of selected retinoic acid response genes such as COUP-TFII, GGTP, THSP-1, GLUT1, and EGR in AVM by qRT-PCR analysis and the expression of these genes are represented by fold change calculated from 10 different AVM nidus samples and 10 number of control tissues (*P < 0.05). The GAPDH was used as the endogenous control for normalization. c The qRT-PCR analysis for ALDH1A2 in AVM and control samples. The fold change in the gene expression of ALDH1A2 in AVM tissues was compared to that of control tissues, and the endothelial cells isolated from AVM (AVMEC) were compared to endothelial cells isolated from control tissues. Analysis was carried out with 10 AVM and 10 control tissues, and endothelial cells were isolated from 5 AVM samples and 5 control samples. The GAPDH was used as endogenous control for normalization (*P < 0.05). d The Western blot analysis of ALDH1A2 protein for the AVM tissues and control tissues. The bottom immunoblot represents the GAPDH for loading control, probed on the same blot used for ALDH1A2 protein. e The densitometry analysis of ALDH1A2 protein and the relative intensity in AVM tissue compared to control tissues. The ALDH1A2 expression is significantly increased in AVM tissues compared to control tissues (*P < 0.05). The GAPDH is the loading control. AVM (n = 11) and control (n = 7)

Fig. 3

Immunostaining of ALDH1A2 in cerebral AVM nidus and control tissues. a Representative image of immunohistochemical analysis with ALDH1A2 antibody in cerebral AVM and control tissues shows increased expression of ALDH1A2 in AVM blood vessels. In control tissues, there is no expression of ALDH1A2 in the blood vessel. The images were collected with × 20 magnification (scale bar, 100 μm). b The immunohistochemical analysis of cerebral AVM and its associated astrocytes shows increased expression of ALDHA2 protein compared to astrocytes in the control tissue. Image 1 shows × 10 magnification and image 2 shows × 40 magnification (scale bar 100 μm). c The bar plot represents the H score analysis of ALDH1A2 protein expression. There is increased expression of this protein in AVM tissue compared to control (*P < 0.05), AVM (n = 10) and control (n = 10). d Immunofluorescence analysis of cerebral AVM and control tissues with ALDH1A2 shows upregulation in AVM blood vessel (green). In control vessels, there is less expression of ALDH1A2 protein. The Hoechst 33342 (blue) dye is used to counter stain nuclei of the cells. The images were collected using × 60 magnification (scale bar, 10 μm). e The bar plot represents the mean fluorescence intensity of ALDH1A2 in AVM and control blood vessel. There is an increased expression of ALDH1A2 in AVM blood vessel compared to control (*P < 0.05), AVM (n = 10) and control (n = 10)

Fig. 4

Triple immunofluorescence staining analysis for GFAP, Vimentin, and ALDH1A2 in cerebral AVM and control tissues. a Triple immunofluorescence staining with GFAP (blue), Vimentin (green) and ALDH1A2 (red) with cerebral AVM nidus. The immunofluorescence staining with GFAP, Vimentin, and ALDH1A2 reveals high expression of these proteins in astrocytes surrounding the AVM vessel. The GFAP + ALDH1A2 + Vimentin immunofluorescence staining demonstrates the co-localized expression of these three proteins in AVM-associated astrocytes. b Triple immunofluorescence staining with GFAP (blue), Vimentin (green), and ALDH1A2 (red) in control tissue, which reveals the minimal expression of ALDH1A2. The images were collected using × 60 magnification (scale bar, 10 μm). c The bar plot represents the mean fluorescent intensity analysis of GFAP, Vimentin and ALDH1A2 in AVM and control vessel. There is a significantly increased expression of all these proteins in AVM tissue compared to control (*P < 0.05), AVM (n = 10) and control (n = 10)

Fig. 5

CYR61 expression analysis in cerebral AVM nidus and its neighboring astrocytes. a Representative images of immunohistochemistry analysis for CYR61 protein using antibody with cerebral AVM nidus and control tissues. The CYR61 is highly expressed in AVM brain parenchyma (box) and less expressed in AVM blood vessel (arrow). In control tissues, the CYR61 protein is not expressed in brain parenchyma (box) as well as in control blood vessel (arrow). The images were collected using × 20 magnification (scale bar, 100 μm). b The bar plot represents the H score analysis of CYR61 expression in AVM and control tissue. There is an increased expression of this protein in AVM tissues compared to control tissues (**P < 0.01), AVM (n = 10) and control (n = 10). c Photomicrograph representing triple immunofluorescence staining with GFAP (blue), ALDH1A2 (green), and CYR61 (red) in cerebral AVM tissues. Immunofluorescence staining with GFAP, ALDH1A2, and CYR61 reveals high expression of these proteins in astrocytes (box) surrounding the AVM vessel (arrow). The GFAP+ ALDH1A2 + CYR61 triple immunofluorescence staining demonstrates the co-localized expression of these three proteins in AVM-associated astrocytes. d The triple immunofluorescence staining with control tissues shows minimal expression of all the three proteins in astrocytes (box) surrounding the control blood vessel (arrow). e The bar plot represents the mean fluorescent intensity analysis of GFAP, ALDH1A2, and CYR61 in AVM and control vessels. There is an increased expression of these proteins in AVM compared to control tissues (*P < 0.05), AVM (n = 10) and control (n = 10)