20(S)-hydroxycholesterol, a natural allosteric agonist of Smoothened, exerts neuroprotective effects associated with angiogenesis and vascular plasticity following ischemia.

BACKGROUND

The Hedgehog signaling pathway has played a key role promoting angiogenesis and neurogenesis in ischemic stroke. To harness the therapeutic potential of Hedgehog signaling in stroke, we investigated the function and potential clinic applications of 20(S)-hydroxycholesterol, a natural small-molecule agonist of Smoothened in the Hedgehog pathway, with respect to its capacity to promote post-stroke neuroprotection, angiogenesis, and cerebrovascular plasticity.

METHODS

In vitro, hypoxia conditions were simulated in brain microvascular endothelial bEnd.3 cells using an oxygen-glucose deprivation/reoxygenation (OGD/R) model. Cell migration capability was evaluated by scratch assay and transwell assay. Endothelial cell angiogenic capacity was evaluated through tube formation assays. Male C57BL/6 J mice were subjected to middle cerebral artery occlusion (MCAO) to establish an ischemic stroke animal model. Neurological deficits were evaluated using the mNSS method. Cerebral infarct volume was measured using TTC staining. The expression levels of Hedgehog signaling pathway and angiogenesis-related proteins were evaluated by immunofluorescence.

RESULTS

Our findings demonstrated that 20(S)-hydroxycholesterol enhanced cell viability, cell migration and tube formation capacity of bEnd.3 cells under OGD/R treatment. In addition, 20(S)-hydroxycholesterol up-regulated the expression of Smoothened and activated Gli transcription factors activity as well as angiogenesis-related genes including Angpt1, Efna5, and Cd31. Moreover. we discovered that 20(S)-hydroxycholesterol significantly reduced cerebral infarct volume and neurological deficits in MCAO model mice.

CONCLUSIONS

20(S)-hydroxycholesterol activates the Hedgehog signaling pathway in ischemic models. It exhibits neuroprotection functions after stroke associated with neuronal angiogenesis and vascular plasticity. This study highlights the therapeutic potential of 20(S)-hydroxycholesterol as a novel agent for ischemic stroke recovery.