Female-specific neuroprotection after ischemic stroke by vitronectin-focal adhesion kinase inhibition.

We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not male, mice. VTN signals through integrins and downstream focal adhesion kinase (FAK). Here, a two day systemic treatment with a small molecule FAK inhibitor starting 6 h after middle cerebral artery occlusion reduced ipsilateral brain injury size by ∼40-45% at 7 and 14 d, as well as inflammation and motor dysfunction in wild-type female, but not male, mice. FAK inhibition also reduced IL-6 expression in the injured female striatum at 24 h by 62%. Inducible selective gene deletion of FAK in astrocytes also reduced acute IL-6 expression by 72% only in females, and mitigated infarct size by ∼80% and inflammation at 14 d after stroke. Lastly, VTN-/- females had better outcomes, but FAK inhibitor treatment had no additional protective or anti-inflammatory effects. Altogether, this suggests that VTN is detrimental in females primarily through FAK and that FAK inhibition provides neuroprotection (cerebroprotection) by reducing VTN-induced IL-6 expression in astrocytes. Thus, VTN signaling can be targeted to mitigate harmful inflammation with relevance to treatments for women with ischemic stroke, who often have worse outcomes than men.

Blood Vitronectin Induces Detrimental Brain Interleukin-6 and Correlates With Outcomes After Stroke Only in Female Mice.

Background and Purpose- Women have worse stroke outcomes than men, especially after menopause. Few studies have focused on female-specific mechanisms, other than hormones. We investigated the role of the blood protein VTN (vitronectin) after ischemic stroke in mice. Methods- Adult male and female VTN knockout and wild-type littermates and C57BL/6 mice received a middle cerebral artery occlusion and the injured brain tissue analyzed 24 hours to 3 weeks later for cell loss and inflammation, as well as neurological function. Blood VTN levels were measured before and after stroke. Results- Intravenously injected VTN leaked extensively from bloodstream into brain infarct and penumbra by 24 hours after stroke. Strikingly, VTN was detrimental in female, but not male, mice, as shown by reduced brain injury (26.2±2.6% versus 13.4±3.8%; P=0.018; n=6 and 5) and forelimb dysfunction in female VTN knockout mice. Stroke increased plasma VTN 2- to 8-fold at 24 hours in females (36±4 versus 145±24 µg/mL; P<0.0001; n=10 and 7), but not males (62±8 versus 68±6; P>0.99; n=10 and 7), and returned to control levels by 7 days. Individually variable VTN levels at 24 hours correlated with stroke-induced brain injury at 7 days only in females. VTN promoted stroke-induced microglia/macrophage activation and leukocyte infiltration in females. Proinflammatory IL (interleukin)-6 greatly increased in the striatum at 24 hours in wild-type mice but was increased ≈60% less in female (739±159 versus 268±111; P=0.02; n=7 and 6), but not male (889±178 versus 1179±295; P=0.73; n=10 and 11), knockout mice. In individual wild-type females, plasma VTN levels correlated with striatal IL-6 expression at 24 hours. The female-specific effect of VTN-induced IL-6 expression following stroke was not due to gonadal hormones, as shown by ovariectomy and castration. Lastly, intrastriatal injection of IL-6 in female mice immediately before stroke reversed the VTN knockout phenotypes of reduced brain injury and microglia/macrophage activation. Conclusions- VTN plays a novel sexually dimorphic detrimental pathophysiological role in females and might ultimately be a therapeutic target to improve stroke outcomes in women.

Vitronectin mitigates stroke-increased neurogenesis only in female mice and through FAK-regulated IL-6.

Vitronectin (VTN) is a blood protein produced mainly by the liver. We show that VTN leaks from the bloodstream into the injury site and neighboring subventricular zone (SVZ) following ischemic stroke (middle cerebral artery occlusion, MCAO) in adult mice. MCAO is known to increase neurogenesis after stroke. VTN inhibits this response in females, but not in males, as shown by ~70% more stroke-induced SVZ neurogenesis in female VTN-/- mice at 14 d. In female VTN-/- mice, stroke-induced expression of interleukin-6 (IL-6) at 24 h was reduced in the SVZ. The closely related leukemia inhibitory factor (LIF) or pro-neurogenic ciliary neurotrophic factor (CNTF) were not affected. The female-specific effect of VTN on IL-6 expression was not due to sex hormones, as shown by ovariectomy and castration. IL-6 injection next to the SVZ reversed the MCAO-induced increase in neurogenesis seen in VTN-/- mice. Our in vitro and vivo data suggest that plasma VTN activates focal adhesion kinase (FAK) in the SVZ following MCAO, which reduces IL-6 expression in astrocytes but increases it in other cells such as microglia/macrophages. Inducible conditional astrocytic FAK deletion increased MCAO-induced IL-6 expression in females at 24 h and blocked MCAO-induced neurogenesis at 14 d, confirming a key detrimental role of IL-6. Collectively, these data suggest that leakage of VTN into the SVZ reduces the neurogenic response to stroke in female mice by promoting IL-6 expression. Reducing VTN or VTN signaling may be an approach to promote neurogenesis for neuroprotection and cell replacement after stroke in females.

Ciliary neurotrophic factor is a key sex-specific regulator of depressive-like behavior in mice.

Ciliary neurotrophic factor (CNTF) is produced by astrocytes and promotes neurogenesis and neuroprotection. Little is known about the role of CNTF in affective behavior. We investigated whether CNTF affects depressive- and anxiety-like behavior in adult mice as tested in the forced swim, sucrose preference and elevated-T maze tests. Female wild type CNTF+/+ mice more readily developed behavioral despair with increased immobility time and decreased latency to immobility in the forced swim test than male CNTF+/+ littermates. The lack of CNTF in CNTF-/- mice had an opposite effect on depressive-like behavior in female mice (reduced immobility time and increased sucrose preference) vs. male mice (increased immobility time). Female wildtype mice expressed more CNTF in the amygdala than male mice. Ovariectomy increased CNTF expression, as well as immobility time, which was significantly reduced in CNTF-/- mice, suggesting that CNTF mediates overiectomy-induced immobility time, possibly in the amygdala. Progesterone but not 17-ß estradiol inhibited CNTF expression in cultured C6 astroglioma cells. Progesterone treatment also reduced CNTF expression in the amygdala and decreased immobility time in female CNTF+/+ but not in CNTF-/- mice. Castration did not alter CNTF expression in males nor their behavior. Lastly, there were no effects of CNTF on the elevated T-maze, a behavioral test of anxiety, suggesting that a different mechanism may underlie anxiety-like behavior. This study reveals a novel CNTF-mediated mechanism in stress-induced depressive-like behavior and points to opportunities for sex-specific treatments for depression, e.g. progesterone in females and CNTF-stimulating drugs in males.

Vitronectin from brain pericytes promotes adult forebrain neurogenesis by stimulating CNTF.

Vitronectin (VTN) is a glycoprotein in the blood and affects hemostasis. VTN is also present in the extracellular matrix of various organs but little is known about its function in healthy adult tissues. We show, in adult mice, that VTN is uniquely expressed by approximately half of the pericytes of subventricular zone (SVZ) where neurogenesis continues throughout life. Intracerebral VTN antibody injection or VTN knockout reduced neurogenesis as well as expression of pro-neurogenic CNTF, and anti-neurogenic LIF and IL-6. Conversely, injections of VTN, or plasma from VTN+/+, but not VTN-/- mice, increased these cytokines. VTN promoted SVZ neurogenesis when LIF and IL-6 were suppressed by co-administration of a gp130 inhibitor. Unexpectedly, VTN inhibited FAK signaling and VTN-/- mice had increased FAK signaling in the SVZ. Further, an FAK inhibitor or VTN increased CNTF expression, but not in conditional astrocytic FAK knockout mice, suggesting that VTN increases CNTF through FAK inhibition in astrocytes. These results identify a novel role of pericyte-derived VTN in the brain, where it regulates SVZ neurogenesis through co-expression of CNTF, LIF and IL-6. VTN-integrin-FAK and gp130 signaling may provide novel targets to induce neurogenesis for cell replacement therapies.