Introduction: Lacunar strokes are both common and disabling; they cause up to 52 strokes per 100,000 people per year and 29-46% of survivors are disabled. Lacunar stroke is part of the spectrum of small vessel disease (SVD) which also causes cognitive impairment and gait disturbance; together these lead to dementia, falls and disability. Current evidence suggests that SVD is caused by a separate aetiology from large vessel stroke, which may be mediated by blood brain barrier (BBB) permeability and may affect organs other than the brain. We set out to establish whether SVD is a multi-system disorder of primary endothelial function, with leakage of blood-brain barrier leading to lacunar stroke, disability, and cognitive impairment. Methods: We recruited 264 patients with a lacunar or cortical stroke (118 lacunar, 146 cortical). All patients received baseline assessment of clinical features, magnetic resonance imaging (MRI), renal function, and assessment of dietary salt. At 1-3 months post-stroke we carried out cognitive testing and contrast MRI to assess blood-brain barrier integrity. We followed patients up at 12 months post-stroke with repeat cognitive testing, MRI, and assessment of disability and recurrent stroke. Results: We established that lacunar stroke has a different risk factor profile to cortical stroke, confirming findings from previous cohorts, but adding dietary salt as a risk factor for lacunar stroke and other SVD features. We confirmed that patients with a clinical stroke who did not have a lesion on diffusion-weighted MRI had the same clinical outcomes at 1 year post-stroke as those patients who did have a lesion. We established that patients who have a lacunar stroke are at as high a risk of post-stroke cognitive impairment as those with a cortical stroke. We found that blood brain barrier leakage predicted cognitive impairment at one year after lacunar and cortical stroke. We established the rates of disability and cognitive impairment at one-year post-lacunar stroke to estimate the required sample size for future trials. Conclusions: Taken together these findings confirm that lacunar stroke is part of a syndrome separate to large vessel stroke and may be mediated through blood brain barrier leakage. Dietary salt is an additional risk factor. The findings support further randomised controlled trials of treatments aimed specifically at lacunar stroke and lifestyle interventions including dietary salt reduction.

Cytochrome P450 3A isozymes (CYP3A) metabolize approximately 50% of therapeutic drugs. It has recently been discovered that human CYP3A mRNA levels can be induced by hypertonicity; a physiological state not previously linked to its regulation. The osmosensitive transcription factor, Nuclear Factor of Activated T-Cells 5 (NFAT5), regulates multiple genes that restore osmolyte homeostasis and promote cell protection during osmotic stress. In silico examinations and in vitro experiments using reporters, knockdown and binding assays in the human intestinal cell line C2bbe1 have revealed an active tonicity-responsive enhancer (TonE) within CYP3A7 intron (+5417/+5427 from CYP3A7 transcriptional start site) that is responsible for NFAT5 binding and NFAT5-dependent regulation of CYP3A isoforms. In addition, hypertonicity-mediated CYP3A induction is also observed in both hepatic and intestinal cell lines. Effects of tonicity changes on in vivo CYP3A expression and function were examined in a humanized CYP3A transgenic mouse with similar tissue expression in humans. More specifically, intervention with prolonged dehydration involving alternating between 24-hour cycles of water-deprivation and water ad lib for 1 week (cyclic water-deprivation; four 24-hour water-deprivation and three 24-hour water ad lib periods), increased expression of NFAT5 target genes Slc6a12 in the liver and kidney (2.5 ± 0.6-fold over water ad lib, n = 14, p = 0.04; and 3.1 ± 0.6-fold, n = 10, p = 0.02, respectively), Akr1b3 in the liver, and Slc5a3 in the kidney. Immunofluorescent microscopy revealed an increase of nuclear-distributed mouse NFAT5 in cyclic water-deprived animals, consistent with NFAT5 activation. Most importantly, CYP3A4 mRNA levels were noted to be elevated in the liver and kidney (11.8 ± 4.8-fold over water ad lib, n = 14, p = 0.04 and 2.2 ± 0.4-fold, n = 9, p = 0.02, respectively), with concurrent CYP3A protein and activity increase. Localized hypertonic environment in the gut was simulated by providing animals with a week-long high-salt diet. The effects of high-salt diet in the gut were similar to those of cyclic water-deprivation in the liver and kidney; where NFAT5 showed nuclear distribution and NFAT5 target gene expression (Slc6a12; 20.5 ± 6.7-fold over a week-long low-salt diet, n = 8, p = 0.02 and Slc6a6; 3.2 ± 0.7-fold, n = 10, p < 0.01, in the duodenum). Furthermore, an increase of CYP3A4 mRNA was observed (2.6 ± 0.5-fold over a week-long low-salt diet, n = 14, p = 0.03), with a corresponding rise in protein expression and activity levels. In summary, increased expression of in vitro and in vivo human CYP3A was achieved using a hypertonic stimulus; concurrent NFAT5 activation and NFAT5 target gene expression were observed. These results suggested a possible binding of activated NFAT5 to CYP3A TonE situated within the intronic region of CYP3A7. It could be further concluded that NFAT5 may be responsible for the hypertonic induction of human CYP3A.

Cytochrome P450 3A isozymes (CYP3A) metabolize approximately 50% of therapeutic drugs. It has recently been discovered that human CYP3A mRNA levels can be induced by hypertonicity; a physiological state not previously linked to its regulation. The osmosensitive transcription factor, Nuclear Factor of Activated T-Cells 5 (NFAT5), regulates multiple genes that restore osmolyte homeostasis and promote cell protection during osmotic stress. In silico examinations and in vitro experiments using reporters, knockdown and binding assays in the human intestinal cell line C2bbe1 have revealed an active tonicity-responsive enhancer (TonE) within CYP3A7 intron (+5417/+5427 from CYP3A7 transcriptional start site) that is responsible for NFAT5 binding and NFAT5-dependent regulation of CYP3A isoforms. In addition, hypertonicity-mediated CYP3A induction is also observed in both hepatic and intestinal cell lines. Effects of tonicity changes on in vivo CYP3A expression and function were examined in a humanized CYP3A transgenic mouse with similar tissue expression in humans. More specifically, intervention with prolonged dehydration involving alternating between 24-hour cycles of water-deprivation and water ad lib for 1 week (cyclic water-deprivation; four 24-hour water-deprivation and three 24-hour water ad lib periods), increased expression of NFAT5 target genes Slc6a12 in the liver and kidney (2.5 ± 0.6-fold over water ad lib, n = 14, p = 0.04; and 3.1 ± 0.6-fold, n = 10, p = 0.02, respectively), Akr1b3 in the liver, and Slc5a3 in the kidney. Immunofluorescent microscopy revealed an increase of nuclear-distributed mouse NFAT5 in cyclic water-deprived animals, consistent with NFAT5 activation. Most importantly, CYP3A4 mRNA levels were noted to be elevated in the liver and kidney (11.8 ± 4.8-fold over water ad lib, n = 14, p = 0.04 and 2.2 ± 0.4-fold, n = 9, p = 0.02, respectively), with concurrent CYP3A protein and activity increase. Localized hypertonic environment in the gut was simulated by providing animals with a week-long high-salt diet. The effects of high-salt diet in the gut were similar to those of cyclic water-deprivation in the liver and kidney; where NFAT5 showed nuclear distribution and NFAT5 target gene expression (Slc6a12; 20.5 ± 6.7-fold over a week-long low-salt diet, n = 8, p = 0.02 and Slc6a6; 3.2 ± 0.7-fold, n = 10, p < 0.01, in the duodenum). Furthermore, an increase of CYP3A4 mRNA was observed (2.6 ± 0.5-fold over a week-long low-salt diet, n = 14, p = 0.03), with a corresponding rise in protein expression and activity levels. In summary, increased expression of in vitro and in vivo human CYP3A was achieved using a hypertonic stimulus; concurrent NFAT5 activation and NFAT5 target gene expression were observed. These results suggested a possible binding of activated NFAT5 to CYP3A TonE situated within the intronic region of CYP3A7. It could be further concluded that NFAT5 may be responsible for the hypertonic induction of human CYP3A.