(T1130-04-23) Regulation of VCAM-1 Expression by the Gut Microbial Metabolite Butyrate: Insights into Molecular Mechanisms

Purpose: Alzheimer’s disease is a progressive neurodegenerative disease characterized by cognitive decline and neuronal loss¹. While amyloid beta (Aβ) peptides and intraneuronal tangles are recognized as major pathological hallmarks² of AD, about 80% of patients also exhibit cerebrovascular pathology<³. This vascular pathology has emerged as a core feature of Alzheimer’s disease that may be detectable before the onset of symptoms. Increased expression of vascular cell adhesion molecule (VCAM)-1, a cerebrovascular inflammatory marker expressed on the blood-brain barrier (BBB) endothelial cells, contributes to cerebrovascular inflammation, which may, in part, be regulated by soluble Aβ exposure to the BBB endothelium. t has been recently discovered that alteration in the gut microbiota⁴ composition is one of the earliest risk factors for Alzheimer’s disease. Changes in the gut microbiota, characterized by reduced levels of anti-inflammatory short-chain fatty acid (SCFA) microbial metabolites like butyrate (NaB), propionate, and acetate⁵ may increase intestinal permeability, which promotes the release on inflammatory cytokines such as TNF alpha. This cytokine can then enter the bloodstream, potentially inducing inflammation in the brain^6,7. However, the mechanistic interrelationship between gut dysbiosis (disruption of gut microbiome), and BBB inflammation in Alzheimer’s disease remains poorly understood.

Methods: The impact of soluble Aβ42 on VCAM-1 expression in human cerebral microvascular endothelial cells (hCMEC/D3) both with and without pre-treatment with NaB. The polarized endothelial monolayer was pre-treated with NaB (10 mM) for 24 hours, followed by treatment with Aβ42 (1 mM) for 3 hours or TNF alpha (10 ng/mL) for 8 hours. We conducted immunocytochemistry or western blot to analyze VCAM-1 expression. To elucidate the underlying molecular mechanisms driving the observed changes in VCAM-1 expression, we investigated the alterations in the expression of phosphorylated Stat3 (Tyr 705) and GATA6 in hCMEC/D3 monolayers using western blots after pre-treatment with NaB and stimulation with Aβ42 or TNF alpha. Subsequently, we examined the contributions of Stat3 and GATA6 in regulating the expression of VCAM-1 by pretreating hCMEC/D3 monolayers with a Stat3 inhibitor (Cryptothanshinone) or by knocking out GATA6 using siRNA. We then assessed changes in VCAM-1 expression following treatment with soluble Aβ42 or TNF alpha using western blot or immunocytochemistry, and the immunocytochemistry samples were imaged using Nikon A1Rsi HD confocal microscope. In addition, to explore the influence of butyrate on VCAM-1 expression, female BL6 mice at 5 weeks of age were colonized with either Clostridium senegalense DSM 25507 WT (butyrate-producing bacteria), or the double-KO mutant (no butyrate-producing bacteria), which was knocked out of KamD (gene in the lysine pathway for butyrate production) and crotonase (gene in the acetyl-CoA pathway for butyrate production) genes. Brains from these mice were harvested, and formalin-fixed-paraffin-embedded blocks were prepared. Immunohistochemistry studies were performed on the mouse brain slices to investigate VCAM-1 expression.

Results: In this study, we investigated the influence of NaB on VCAM-1 expression following stimulation with Aβ42 or TNF alpha, where TNF alpha served as a positive control. The findings of the current study indicate that exposure to soluble Aβ42 or TNF alpha resulted in an increase in VCAM-1 expression in hCMEC/D3 cell monolayers; the addition of NaB was found to counteract with this increase in VCAM-1 expression. Additionally, exposure to soluble Aβ42 or TNF alpha activated the inflammatory signaling pathway, marked by a significant increase in the expressions of GATA6 and phosphorylated Stat3 (Tyr 705). Moreover, the decrease in VCAM-1 expression observed after pre-treatment with NaB correlated with a reduction in the phosphorylation of Stat3 (Tyr 705) triggered by soluble Aβ42 or TNF alpha. Furthermore, inhibiting Stat3 or knocking out GATA6 using siRNA reduced the soluble Aβ42/TNF alpha-mediated increase in VCAM-1 expression (Figure 2). Immunohistochemistry studies showed that mice colonized with butyrate- producing bacteria exhibited lower VCAM-1 expression in the cortex as compared to control mice (Figure 3).

Conclusion: In summary, the current study demonstrated that Aβ42 increased the expression of VCAM-1 on the BBB endothelium. The NaB treatment decreased Aβ42 triggered increase in VCAM-1, which may be mediated by stimulating Stat3/GATA6 signaling pathway to reduce Aβ42-mediated VCAM-1 expression (Figure 1C).

References: 1. Rainulf a. Stelzmann HNS, and F. Reed Murtagh. An English Translation of Alzheimer’s 1907 Paper, “Uber eine eigenartige Erkankung der Hirnrinde”. Clinical Anatomy Web site. https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ca.980080612. Published 1995. Accessed March 2021.
2. Jack CR, Jr., Knopman DS, Jagust WJ, et al. Hypothetical model of dynamic biomarkers of the Alzheimer's pathological cascade. Lancet Neurol. 2010;9(1):119-128.
3. Scheibel AB. Alterations of the cerebral capillary bed in the senile dementia of Alzheimer. Ital J Neurol Sci. 1987;8(5):457-463.
4. Vinolo MA, Rodrigues HG, Nachbar RT, Curi R. Regulation of inflammation by short chain fatty acids. Nutrients. 2011;3(10):858-876.
5. Cattaneo A, Cattane N, Galluzzi S, et al. Association of brain amyloidosis with pro-inflammatory gut bacterial taxa and peripheral inflammation markers in cognitively impaired elderly. Neurobiol Aging. 2017;49:60-68.
6. Braniste V, Al-Asmakh M, Kowal C, et al. The gut microbiota influences blood-brain barrier permeability in mice. Sci Transl Med. 2014;6(263):263ra158.
7. Zuliani G, Ranzini M, Guerra G, et al. Plasma cytokines profile in older subjects with late onset Alzheimer's disease or vascular dementia. J Psychiatr Res. Oct 2007;41(8):686-93. doi:10.1016/j.jpsychires.2006.02.008


Sodium butyrate decreases Aβ42 and TNF alpha mediated increase in VCAM-1 expression. Experimental scheme. The hCMEC/D3 monolayers were cultured on 35 mm glass round bottom dishes or 6 well plates in D3 media. Upon reaching confluence, cells were pretreated with/without sodium butyrate (10 uM) for 21 hrs., followed by treatment with/without Aβ42 (1 uM) or TNF alpha (10 ng/mL) for 8hrs. (A) Confocal micrographs depict Aβ42 stimulated an increase in the binding of AF-647 labelled VCAM-1 IgG to the luminal surface of hCMEC/D3 monolayers, while sodium butyrate reverses Aβ42 and TNF alpha’s effects, decreasing VCAM-1 expression. AF-647-VCAM-1 IgG is stained red, and the nuclei are stained blue (DAPI). Scale bar = 20 um. (B) Immunoblots demonstrate butyrate mediated reduction in VCAM-1 expression following TNF alpha stimulation. Quantification by densitometry. Data expressed as mean ± S.D. ****p < 0.0001, *p < 0.05; determined by Student’s t-test. (C) Aβ42-induced activation of Stat3 promotes its phosphorylation at the tyrosine 705 site. Subsequently, this phosphorylation event activates GATA6, a known inducer of VCAM-1 expression. Butyrate exerts an inhibitory effect on the increase in VCAM-1 expression by suppressing Stat3 activation. Consequently, this inhibition downregulates GATA6 expression, leading to a reduction in VCAM-1 expression.



Phospho-STAT3 inhibitor, cryptotanshinone and GATA6 KO decreases Aβ42 and TNF alpha mediated increase in VCAM-1 expression. Experimental scheme. The hCMEC/D3 monolayers were pretreated with/without cryptotanshinone (40 M) for 1 hr. or with GATA6 siRNA for 6 hrs. followed by treatment with/without Aβ42 (1 M) for 3hrs or TNF alpha (20 ng/mL) for 8 hrs. (A&B) Confocal micrographs and immunoblots depict Aβ42 and TNF alpha stimulated an increase in the expression VCAM-1 in hCMEC/D3 monolayers, while cryptothanshinone and GATA6 KO reverses Aβ42 and TNF alpha’s effects, decreasing VCAM-1 expression. (A) AF-647-VCAM-1 IgG is stained red, and the nuclei are stained blue (DAPI). Scale bar = 20 m. (B) Data expressed as mean ± S.D. **p < 0.001, *p < 0.05; determined by Student’s t-test



Germ-free mice colonized with butyrate-producing bacteria exhibit a reduction in VCAM-1 expression as demonstrated by immunohistochemistry performed on mice brain.