Neuroprotective effects respond to cerebral ischemia without susceptibility to HB-tumorigenesis in VHL heterozygous knockout mice

Abstract

The von Hippel-Lindau (VHL) tumor suppressor gene plays a prominent role in the development of hemangioblastomas (HBs) within specific regions of the human' central nervous system (CNS). Alterations in VHL gene are rarely observed in the more common features of human VHL-related tumors in animal models, and VHL heterozygous knockout (VHL+/-) mice do not develop HBs. We tested whether VHL heterozygous knockout mice exhibited genetic predisposition to the development of HBs and conferred a selective advantage involving growth of blood vessels to its carrier. No differences were observed between wild-type and VHL+/- mice in development ad reproduction. The heterozygous VHL+/- mice did not develop higher genetic susceptibility to CNS-HBs over their lifetime. Furthermore, this recessive VHL gene heterozygosity is relatively stable. Interestingly, we found these heterozygous VHL+/- mice gained an advantage conferring to angiogenic ability in a particular environment, compared with wild-type mice. The heterozygous VHL+/- mice obviously enhanced hypoxia inducible factor-1 (HIF)-dependent and Twist1 angiogenic mechanism in response to acute cerebral ischemia, resulting in decreased cerebral tissue damage and neuroprotective response through neovascularization. Our findings provide evidence of partial loss function of VHL as a novel precise therapeutic target in acute cerebral ischemia.

Keywords: cerebral ischaemia; gene therapy; hemangioblastoma; neuroprotection; von Hippel-Lindau gene (VHL).

Figure 1. Production of VHL heterozygous knockout mice by TALEN-mediated gene targeting

(A) Double-stranded DNA sequence of the VHL locus that was targeted with TALENs. The TALEN binding sites are highlighted in blue and the spacer region is underlined. (B) Diagram of VHL Exon 1 (yellow oval) showing the start of translation (ATG) and the TALEN binding sites (arrows, starting at 169 bp and ending at 286 bp). (C) DNA sequence of the wild type (WT) VHL with TALEN binding sites. The underlined sequence indicates high sensitive region for mutation. Immediately beneath the wild type sequence is the sequence derived from cloned PCR products from founder (F0) mice (Mut), which demonstrates GG deletion, GCC deletion, CG insertion (highlight in red), GCGGCC deletion and GCC deletion. (D) DNA sequence chromatograms of F1 RT-PCR products demonstrating the GG deletion in the VHL+/− sample. (E, F) Relative VHL mRNA/protein level by qRT-PCR or Westernblot from cerebral cortex of wild type (+/+) and heterozygous (VHL+/−) mice. *P<0.05, **P<0.01 compared to the wild type mice.

Figure 2. VHL deletion shows decreased severity of cerebral ischemia in mice

Compared to the wild type mice, VHL heterozygous (+/−) knockout mice showed significantly lower modified neurological severity scores (mNSS) (A), lower limb placing score (B) and lower elevated body swing test (C) following MCAO (n = 12 per group). *P<0.05, **P<0.01 compared to the wide type mice. (D) At day 7 after MCAO, brain slices were stained with 2, 3, 5-triphenyltetrazolium chloride (TTC), and representative photographs were shown. The healthy tissue was stained with red and the infracted tissue was unstained and showed white. (E) Compared to the wild type mice, VHL heterozygous (+/−) knockout mice show significantly reduced infarct volume at 7 days after MCAO. *P<0.05, **P<0.01. d: days. MCAO: Middle cerebral artery occlusion.

Figure 3. VHL heterozygous deletion increases the expression of HIF-1α and its downstream genes, and Twist 1 gene at 7 days after MCAO

(A) Compared to the wild type mice with MCAO, VHL heterozygous (+/−) knockout mice showed unregulated HIF-1α mRNA level, and upregulated expression in its downstream genes EPO (B) and VEGF (C) by qRT-PCR. (D) Representative images of western blot in HIF-1α and Twist 1 protein. (E) Quantitative data of western blot demonstrated increased HIF-1α protein (n = 8 per group). Quantitative analysis also showed increased Twist 1 mRNA level (F) and level (G) in heterozygous (+/−) knockout mice, compared with the wild type mice after MCAO. Data were expressed as mean values ± standard deviation (SD). *P<0.05, **P<0.01. MCAO: Middle cerebral artery occlusion. There are no difference between the sham-WT group and the sham-VHL+/− group.

Figure 4. VHL heterozygous deletion showed neuroprotective effects in mice with cerebral ischemia

(A) Immunohistochemical staining for Caspase-3 and GFAP in IBZ on 7 days following MCAO. (B) Quantitative data of Caspase-3 and GFAP levels by immunohistochemical staining. Compared to the wild type mice with MCAO, VHL heterozygous (+/−) knockout mice showed significantly lower Caspase-3 and higher GFAP levels. (C) Quantitative data of p-AKT and p-GSK-3β proteins by western blot (n = 8 per group). Compared to the wild type mice with MCAO, VHL heterozygous (+/−) knockout mice show significantly increased p-AKT (D) and p-GSK-3β proteins (E) 7 days following MCAO. *P<0.05, **P<0.01. MCAO: Middle cerebral artery occlusion. There are no difference between the sham-WT group and the sham-VHL+/− group.