Protective Role of Short-Chain Fatty Acids against Ang- II-Induced Mitochondrial Dysfunction in Brain Endothelial Cells: A Potential Role of Heme Oxygenase 2

Objectives: Short-chain fatty acids (SCFAs), the main metabolites released from the gut microbiota, are altered during hypertension and obesity. SCFAs play a beneficial role in the cardiovascular system. However, the effect of SCFAs on cerebrovascular endothelial cells is yet to be uncovered. In this study, we use brain endothelial cells to investigate the in vitro effect of SCFAs on heme oxygenase 2 (HO-2) and mitochondrial function after angiotensin II (Ang-II) treatment. Methods: Brain human microvascular endothelial cells were treated with Ang-II (500 nM for 24 h) in the presence and absence of an SCFAs cocktail (1 μM; acetate, propionate, and butyrate) and/or HO-2 inhibitor (SnPP 5 μM). At the end of the treatment, HO-2, endothelial markers (p-eNOS and NO production), inflammatory markers (TNFα, NFκB-p50, and -p65), calcium homeostasis, mitochondrial membrane potential, mitochondrial ROS and H2O2, and mitochondrial respiration were determined in all groups of treated cells. Key Results: Our data showed that SCFAs rescued HO-2 after Ang-II treatment. Additionally, SCFAs rescued Ang-II-induced eNOS reduction and mitochondrial membrane potential impairment and mitochondrial respiration damage. On the other hand, SCFAs reduced Ang-II-induced inflammation, calcium dysregulation, mitochondrial ROS, and H2O2. All of the beneficial effects of SCFAs on endothelial cells and mitochondrial function occurred through HO-2. Conclusions: SCFAs treatment restored endothelial cells and mitochondrial function following Ang-II-induced oxidative stress. SCFAs exert these beneficial effects by acting on HO-2. Our results are opening the door for more studies to investigate the effect the of SCFAs/HO-2 axis on hypertension and obesity-induced cerebrovascular diseases.


Introduction
Mitochondria play an important role in cellular respiration, cell death, the regulation of innate immunity, and calcium homeostasis, and are crucial in regulating brain microvascular function and cerebral blood flow [1,2]. Interestingly, the mitochondria-mediated

Protein Expression
Western blot analysis for HO-2, t-and p-eNOS was performed in cell lysates as previously described [17,18]. Briefly, the cells were harvested, lysed in RIPA buffer supplemented with proteinase and phosphatase inhibitors, and then sonicated using a sonicator. After centrifugation for 10 min at 10,000 rpm, the total protein was quantified using the BCA assay and 20 ug of protein per sample were loaded into SDS-PAGE gels. The proteins were then transferred to PVDF membranes, incubated in 5% milk, and incubated with primary antibodies for HO-2 (1:1000 dilution), total-and phosphorylated eNOS (1:1000 dilution). GAPDH (1:5000) was used as a loading control.

Measurement of Ca 2+ Uptake by Mitochondria
Ratiometric measurements of [Ca 2+ ] in mitochondria were performed in HBMECs using mitochondrial Ca 2+ adenovirus ( mt )Pericam (Admt Pericam), as previously described [19]. Pericam fluorescence was detected using a customized Nikon Eclipse Ti2 inverted light microscope. Pericam was excited at 405 nm and 480 nm, and its emission was recorded at 535 nm. Real-time Pericam fluorescence ratios were recorded before and after plateletderived growth factor (PDGF) was added (20 ng/mL) and were quantified using ImageJ. The summary data represent the average difference in basal mitochondrial [Ca 2+ ].

Measurement of Cytosolic Ca 2+
Calcium release into the cytosol was measured, as previously described [20]. Briefly, pretreated primary HBMECs were loaded with 20 µM Fluo-4 for 15 min. Cells were then washed with isotonic buffer and the assay was performed in a buffer solution. Fluo-4 fluorescence was determined using a fluorescence microscope.

Measurement of Mitochondrial ROS Production
Mitochondrial ROS production was measured in live cultured HBMECs using the dihydroethidium derivative MitoSOX Red. Following treatments, cultured cells were rinsed in warm HBSS buffer and then loaded with MitoSOX Red (5 µM) and MitoTracker Green FM (1 µM) [21] diluted in HBSS buffer for 20 min at 37 • C. The cells were then rinsed in warm HBSS buffer, imaged using a fluorescence microscope, and analyzed using NIH ImageJ. The data are presented as the ratio of integrated density MitoSOX Red signal to MitoTracker Green FM signal. Cells were treated with Rotenone (1 µM) for 1 h and were Antioxidants 2023, 12, 160 4 of 14 used as a positive control. Other cultured HBMECs were pretreated with a mitochondrial ROS scavenger (mitoTEMPO, 10 µM) in the absence or presence of Ang-II. The cells were then washed and loaded with MitoTracker and MitoSOX following the same protocol as above.

Measurement of Cellular Hydrogen Peroxide
To assess the H 2 O 2 release in response to Ang-II in the presence or absence of SCFAs, extracellular H 2 O 2 levels were measured using the fluorescent probe Amplex Red (Molecular Probes, ThermoFisher) [22] following the manufacturer's instructions. Briefly, following treatment, cultured HBMECs were exposed to Amplex Red (2 mmol/L) diluted in the appropriate buffer. The supernatant was then collected and a volume of 100 µl was loaded into a 96-well plate and fluorescence was measured relative to standard controls generated by serial dilutions of H 2 O 2 on a spectrophotometer using excitation and emission levels of 490 nm and 585 nm, respectively. To correct for background fluorescence, measurements were compared to a no-H 2 O 2 control. All fluorescence values were normalized to the total protein from each dish using a BCA protein assay.

Quantification of Nitric Oxide
Nitric Oxide (NO) levels were measured using the fluorescent probe DAF2-DA (Sigma) [23]. Following treatments, cultured HBMECs were washed with warm DPBS and then incubated with a fluorescent nitric oxide probe and DAF2-DA (5 µM in ECM medium) for 60 min. The HBMECs were then rinsed and imaged under a fluorescence microscope. To induce NO release, HBMECs were exposed to PDGF (20 ng/mL) + glutamine (1 µM) [24]. Continuous imaging was performed for 10 min following stimulation. For the negative control measurements, the cells were subjected to LNNA for 30 min, then washed, and then assayed for NO production following the same protocol as above. For the positive control measurements, the DAF2-DA-loaded cells were stimulated with sodium nitroprusside (SNP). The amount of NO produced is expressed as fluorescence intensity normalized to that at baseline.

Bioenergetics by Seahorse
For experiments in the Seahorse XF analyzer (Seahorse Bioscience), HBMECs were plated into 96-well Seahorse V3 PET plates at a density of 50,000 per well 24 h before the treatment. HBMECs were then washed and equilibrated in Seahorse assay medium containing 25 mM glucose, 1 mM pyruvate, and 2 mM L-Glutamine, and subjected to different treatments of Ang-II, SCFAs, and/or HO-2 inhibitor, as indicated above. At 18 h post-treatment, a mitochondrial stress test was performed in a Seahorse Bioscience XF96 analyzer with sequential additions of oligomycin A, FCCP, and antimycin/rotenone at 1, Antioxidants 2023, 12, 160 5 of 14 1.5, and 2 µM each, respectively. The ATP-dependent oxygen consumption rate (OCR) was calculated by subtracting the OCR after the addition of oligomycin A from the baseline OCR and the basal extracellular acidification rate (ECAR) was measured prior to the addition of glucose.

Statistical Analysis
Data are expressed as mean ± SEM and were analyzed using GraphPad Prism 9.0 software. All data sets were analyzed for normality and equal variance. Kruskal-Wallis test and Dunn's post hoc test were used for data sets where normal distribution could not be assumed. Two-tailed unpaired Student's t-test and one-way ANOVA, followed by Tukey's multiple comparison tests, were used for data sets with normal distribution. Two-way ANOVA followed by Tukey's multiple comparison tests were used for grouped data sets. A p-value <0.05 was considered significant.

SCFAs Reverse Ang-II-Induced Downregulation of HO-2
To examine whether SCFAs regulate HO-2 during Ang-II treatments, we analyzed the HO-2 expression level and activity in HBMECs subjected to Ang-II in the presence or absence of SCFAs. Decreased levels of HO-2 expression were observed in HBMECs following Ang-II treatment compared to the vehicle ( Figure 1A,B). Interestingly, in the presence of SCFAs, HBMECs exhibit fully restored HO-2 expression when compared to vehicle-treated cells ( Figure 1A,B). Next, we tested the activity of HO-2 by measuring the bilirubin levels. Ang-II significantly decreased bilirubin levels which were recovered with SCFAs co-treatment ( Figure 1C,D). These results suggest that SCFAs exert a regulatory effect on HO-2 expression and activity. plated into 96-well Seahorse V3 PET plates at a density of 50,000 per well 24 hours before the treatment. HBMECs were then washed and equilibrated in Seahorse assay medium containing 25 mM glucose, 1 mM pyruvate, and 2 mM L-Glutamine, and subjected to different treatments of Ang-II, SCFAs, and/or HO-2 inhibitor, as indicated above. At 18 h post-treatment, a mitochondrial stress test was performed in a Seahorse Bioscience XF96 analyzer with sequential additions of oligomycin A, FCCP, and antimycin/rotenone at 1, 1.5, and 2 μM each, respectively. The ATP-dependent oxygen consumption rate (OCR) was calculated by subtracting the OCR after the addition of oligomycin A from the baseline OCR and the basal extracellular acidification rate (ECAR) was measured prior to the addition of glucose.

Statistical Analysis
Data are expressed as mean ± SEM and were analyzed using GraphPad Prism 9.0 software. All data sets were analyzed for normality and equal variance. Kruskal-Wallis test and Dunn's post hoc test were used for data sets where normal distribution could not be assumed. Two-tailed unpaired Student's t-test and one-way ANOVA, followed by Tukey's multiple comparison tests, were used for data sets with normal distribution. Twoway ANOVA followed by Tukey's multiple comparison tests were used for grouped data sets. A p-value <0.05 was considered significant.

SCFAs Reverse Ang-II-Induced Downregulation of HO-2
To examine whether SCFAs regulate HO-2 during Ang-II treatments, we analyzed the HO-2 expression level and activity in HBMECs subjected to Ang-II in the presence or absence of SCFAs. Decreased levels of HO-2 expression were observed in HBMECs following Ang-II treatment compared to the vehicle ( Figure 1A,B). Interestingly, in the presence of SCFAs, HBMECs exhibit fully restored HO-2 expression when compared to vehicle-treated cells ( Figure 1A,B). Next, we tested the activity of HO-2 by measuring the bilirubin levels. Ang-II significantly decreased bilirubin levels which were recovered with SCFAs co-treatment ( Figure 1C,D). These results suggest that SCFAs exert a regulatory effect on HO-2 expression and activity.  showing that the Ang-II-induced reduction in HO-2 protein expression and activity in brain microvascular endothelial cells was rescued by the SCFAs/HO-2 axis. * p < 0.05; ** p < 0.01; *** p < 0.001. N = 3-4. SCFAs: short-chain fatty acids; Ang II: angiotensin II; and HO-2 I: heme oxygenase 2 inhibitor.

SCFAs Improve Ang-II-Induced Endothelial Dysfunction by Regulating HO-2
To assess the effect of SCFAs on the Ang-II-induced endothelial dysfunction in vitro, HBMECs were exposed to Ang-II in the presence or absence of SCFAs, and the endothelial function markers were evaluated. A significant reduction in phosphorylated eNOS levels (Figure 2A,B) and NO production ( Figure 2C) and an increased level of VCAM1 ( Figure 2D) were observed in Ang-II-treated cells compared to the vehicle. These effects were fully reversed in the presence of SCFAs ( Figure 2). Interestingly, pharmacological inhibition of HO-2 using an HO-2 inhibitor (HO-2 I, SnPP 5 µM) annulled the beneficial effects of SCFAs in vitro ( Figure 2). These data indicate that the SCFAs reverse the Ang-II-induced endothelial dysfunction via an HO-2-mediated pathway.

SCFAs Reduce Ang-II-Induced Endothelial Inflammation by Regulating HO-2
Inflammation plays a detrimental role in regulating ECs and blood flow [25,26]. Along the same lines, the present study showed that HBMECs subjected to Ang-II displayed higher expression levels of TNF, NFB-p50, and NFB-p65 ( Figure 3) compared to vehicle-treated HBMECs. Similarly, co-treatment with SCFAs prevented the increase in these inflammatory markers, an effect that is abolished by the presence of the HO-2 inhibitor, indicating an intermediatory role of HO-2 in the SCFAs-mediated effect ( Figure 3). showing that the Ang-IIinduced reductions in eNOS expression and NO production and increased VCAM1 expression in brain microvascular endothelial cells were reversed by the SCFAs/HO-2 axis. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. N = 3-7. SCFAs: short-chain fatty acids; Ang II: angiotensin II; HO-2 I: heme oxygenase 2 inhibitor; eNOS: nitric oxide synthase; NO: nitric oxide; and VCAM1: vascular cell adhesion molecule.

SCFAs Reduce Ang-II-Induced Endothelial Inflammation by Regulating HO-2
Inflammation plays a detrimental role in regulating ECs and blood flow [25,26]. Along the same lines, the present study showed that HBMECs subjected to Ang-II displayed higher expression levels of TNFα, NFκB-p50, and NFκB-p65 ( Figure 3) compared to vehicletreated HBMECs. Similarly, co-treatment with SCFAs prevented the increase in these inflammatory markers, an effect that is abolished by the presence of the HO-2 inhibitor, indicating an intermediatory role of HO-2 in the SCFAs-mediated effect ( Figure 3).

The SCFAs/HO-2 Axis Regulates Calcium Homeostasis in Mitochondria from Cerebral ECs
HO-2 activity is closely regulated by cellular calcium during neuronal activity [27]. To test whether this relationship exists between the SCFAs/HO-2 axis and mitochondrial calcium homeostasis, we evaluated the cytosolic and mitochondrial calcium levels following exposure to Ang-II and in the presence or absence of SCFAs. At one-day post-Ang-II treatment, HBMECs exhibited increased cytosolic and mitochondrial Ca 2+ levels compared to vehicle-treated cells (Figure 4). Interestingly, SCFAs co-treatment normalized both cy-

The SCFAs/HO-2 Axis Regulates Calcium Homeostasis in Mitochondria from Cerebral ECs
HO-2 activity is closely regulated by cellular calcium during neuronal activity [27]. To test whether this relationship exists between the SCFAs/HO-2 axis and mitochondrial Antioxidants 2023, 12, 160 7 of 14 calcium homeostasis, we evaluated the cytosolic and mitochondrial calcium levels following exposure to Ang-II and in the presence or absence of SCFAs. At one-day post-Ang-II treatment, HBMECs exhibited increased cytosolic and mitochondrial Ca 2+ levels compared to vehicle-treated cells (Figure 4). Interestingly, SCFAs co-treatment normalized both cytosolic and mitochondrial Ca 2+ levels, an effect that was abolished in the presence of HO-2 I (Figure 4). brain microvascular endothelial cells were reversed by the SCFAs/HO-2 axis. **** p < 0.0001. N = 7-8. SCFAs: short-chain fatty acids; Ang II: angiotensin II; HO-2 I: heme oxygenase 2 inhibitor; TNF: tumor necrosis factor-alpha; and p50 and p65: NF-κB subunits.

The SCFAs/HO-2 Axis Regulates Calcium Homeostasis in Mitochondria from Cerebral ECs
HO-2 activity is closely regulated by cellular calcium during neuronal activity [27]. To test whether this relationship exists between the SCFAs/HO-2 axis and mitochondrial calcium homeostasis, we evaluated the cytosolic and mitochondrial calcium levels following exposure to Ang-II and in the presence or absence of SCFAs. At one-day post-Ang-II treatment, HBMECs exhibited increased cytosolic and mitochondrial Ca 2+ levels compared to vehicle-treated cells (Figure 4). Interestingly, SCFAs co-treatment normalized both cytosolic and mitochondrial Ca 2+ levels, an effect that was abolished in the presence of HO-2 I (Figure 4).

SCFAs Normalized Mitochondrial Membrane Potential by Mediating HO-2 following Ang-II Treatment
Mitochondria utilize the electrochemical potential across their inner membrane (∆Ψm) to stimulate mitochondrial Ca 2+ entry and promote mt ROS production, in part, by OXPHOS activity stimulation. In the current study, we investigated whether the activation of the SCFAs/HO-2 axis influenced ∆Ψm following Ang-II treatment. Our data, using the TMRM fluorescence, showed that ∆Ψm was hyperpolarized following Ang-II treatment. While the treatment with only SCFAs was able to preserve membrane potential, the combination with HO-2 I abolished this effect ( Figure 5).

The SCFAs/HO-2 Axis Regulates Mitochondrial ROS, H 2 O 2 and Mitochondrial Function
HO-2 has been shown to possess an antioxidant effect in the cerebrovascular endothelium [28]. To test whether SCFAs will prevent Ang-II-induced mitochondrial oxidative stress via the HO-2 pathway, we measured mitochondrial (mt)ROS production and its byproduct, cellular H 2 O 2 . Ang-II treatment increased the level of Mito-SOX fluorescence intensity, indicating higher mitochondrial ROS production; the co-treatment with SCFAs abolished Ang-II-induced mtROS, whereas the presence of HO-I annulled the positive effect of the SCFAs ( Figure 6A). Since most of the ROS produced by mitochondria are rapidly converted to H 2 O 2 by manganese superoxide dismutase (MnSOD), we measured the levels of H 2 O 2 in cultured HBMECs using the Amplex Red assay. The results showed that the treatment with SCFAs reduced the H 2 O 2 levels induced by the Ang-II treatment ( Figure 6B). This effect was abolished in the presence of the HO-2 inhibitor.
Mitochondria utilize the electrochemical potential across their inner membrane (ΔΨm) to stimulate mitochondrial Ca 2+ entry and promote mtROS production, in part, by OXPHOS activity stimulation. In the current study, we investigated whether the activation of the SCFAs/HO-2 axis influenced ΔΨm following Ang-II treatment. Our data, using the TMRM fluorescence, showed that ΔΨm was hyperpolarized following Ang-II treatment. While the treatment with only SCFAs was able to preserve membrane potential, the combination with HO-2 I abolished this effect ( Figure 5).

The SCFAs/HO-2 Axis Regulates Mitochondrial ROS, H2O2 and Mitochondrial Function
HO-2 has been shown to possess an antioxidant effect in the cerebrovascular endothelium [28]. To test whether SCFAs will prevent Ang-II-induced mitochondrial oxidative stress via the HO-2 pathway, we measured mitochondrial (mt)ROS production and its byproduct, cellular H2O2. Ang-II treatment increased the level of Mito-SOX fluorescence intensity, indicating higher mitochondrial ROS production; the co-treatment with SCFAs abolished Ang-II-induced mtROS, whereas the presence of HO-I annulled the positive effect of the SCFAs ( Figure 6A). Since most of the ROS produced by mitochondria are rapidly converted to H2O2 by manganese superoxide dismutase (MnSOD), we measured the levels of H2O2 in cultured HBMECs using the Amplex Red assay. The results showed that the treatment with SCFAs reduced the H2O2 levels induced by the Ang-II treatment (Figure 6B). This effect was abolished in the presence of the HO-2 inhibitor.

SCFAs Rescued Ang-II-Induced Mitochondrial Respiration Damage by Mediating HO-2
The same pattern was observed in oxygen consumption using a Seahorse stress test ( Figure 7A). While a decreased oxygen consumption rate (OCR) was observed in the presence of Ang-II, HBMECs exhibited an increased extracellular acidification rate (ECAR) following Ang-II treatments (Figure 7B), indicating a metabolic switch to glycolysis. This phenotype was reversed in the presence of SCFAs. Treatment with the HO-2 inhibitor abolished the beneficial effect of SCFAs on OCR levels following Ang-II treatments (Fig-A B

SCFAs Rescued Ang-II-Induced Mitochondrial Respiration Damage by Mediating HO-2
The same pattern was observed in oxygen consumption using a Seahorse stress test ( Figure 7A). While a decreased oxygen consumption rate (OCR) was observed in Antioxidants 2023, 12, 160 9 of 14 the presence of Ang-II, HBMECs exhibited an increased extracellular acidification rate (ECAR) following Ang-II treatments (Figure 7B), indicating a metabolic switch to glycolysis. This phenotype was reversed in the presence of SCFAs. Treatment with the HO-2 inhibitor abolished the beneficial effect of SCFAs on OCR levels following Ang-II treatments (Figure 7). Interestingly, the ECAR levels completely declined in the presence of the HO-2 inhibitor indicating other cytosolic pathways where HO-2 potentially participate to maintain metabolic activity.

Discussion
Overall, this study showed that HO-2 expression and activity are altered during cellular stress. Additionally, the reduction in HO-2 expression and activity in cerebrovascular endothelial cells causes mitochondrial and endothelial dysfunction. SCFAs were able to restore the level of HO-2 and therefore rescued the mitochondrial and endothelial function.
The relationship between HO-1 and Ang-II-induced hypertension has been well documented in the literature [29]. Indeed, HO-1 levels significantly decreased in response to Ang-II, and HO-1 overexpression reversed the detrimental effect of Ang-II on vascular function and blood pressure [29,30]. However, little is known about the relationship between Ang-II and HO-2. HO-2 possesses cytoprotective effects due to its antioxidant, antiapoptotic, and anti-inflammatory effects [15]. Here, we showed evidence that Ang-II was able to reduce the level and activity of HO-2 in primary HBMECs. The current data demonstrate that HO-2 plays an important role in regulating HMBEC function during hypertensive conditions.
Hypertension is associated with reduced levels of SCFAs [31,32]. While several studies demonstrated the protective role of SCFAs during hypertension, via normalizing blood pressure and vascular reactivity [32][33][34], it remains difficult to dissect whether the

Discussion
Overall, this study showed that HO-2 expression and activity are altered during cellular stress. Additionally, the reduction in HO-2 expression and activity in cerebrovascular endothelial cells causes mitochondrial and endothelial dysfunction. SCFAs were able to restore the level of HO-2 and therefore rescued the mitochondrial and endothelial function.
The relationship between HO-1 and Ang-II-induced hypertension has been well documented in the literature [29]. Indeed, HO-1 levels significantly decreased in response to Ang-II, and HO-1 overexpression reversed the detrimental effect of Ang-II on vascular function and blood pressure [29,30]. However, little is known about the relationship between Ang-II and HO-2. HO-2 possesses cytoprotective effects due to its antioxidant, antiapoptotic, and anti-inflammatory effects [15]. Here, we showed evidence that Ang-II was able to reduce the level and activity of HO-2 in primary HBMECs. The current data demonstrate that HO-2 plays an important role in regulating HMBEC function during hypertensive conditions. Hypertension is associated with reduced levels of SCFAs [31,32]. While several studies demonstrated the protective role of SCFAs during hypertension, via normalizing blood pressure and vascular reactivity [32][33][34], it remains difficult to dissect whether the effect of SCFAs on vascular endothelial cells was direct or a consequence of blood pressure reduction. Although there are studies that provide convincing evidence that SCFAs, through a direct or indirect mechanism, can activate HO-1 [35], there have been no studies to date that have evaluated the effect of SCFAs on HO-2. In the present study, we evaluated the direct effect of SCFAs on HBMECs treated with Ang-II in vitro. SCFAs supplementation was able to recover HO-2 expression and activity following Ang-II treatment. Since HO-2 is involved in many cytoprotective pathways [15], we speculate that SCFAs, by acting on HO-2, will positively impact cellular function under stress.
Typically, Ang-II induces endothelial dysfunction by reducing NO and increasing the proinflammatory markers and adhesion molecules [36]. Our data are in accordance with these observations since we showed a reduction in NO production and an increase in inflammatory markers and adhesion molecules in HBMECs following Ang-II exposure. Cotreatment with SCFAs reversed these effects. The beneficial effect of SCFAs on endothelial function such as the increase in NO production [36], anti-inflammatory effects [37,38], and the reduction in adhesion molecules [39] is very well established. However, the exact mechanism by which SCFAs exert this beneficial effect, especially in HBMECs is lacking. In the present study, we have evidence that SCFAs' beneficial effects on HBMECs were achieved through HO-2. The outcome of the study and the data are a proof of concept that the SCFAs/HO-2 axis is a key determinant of endothelial function.
The relationship between HO-1 and mitochondrial function is well-documented [13,14]. However, little is known about the relationship between HO-2 and mitochondrial function, especially in cerebrovascular endothelial cells. A recent study showed that similar to HO-1, HO-2 can translocate to the mitochondria [16]. However, the role of HO-2 in the mitochondria remains largely unknown. The present data shows that HO-2 is a key component for mitochondrial function as it regulates mitochondrial Ca 2+ homeostasis, membrane potential, mitochondrial ROS, H 2 O 2 , and oxygen consumption. Furthermore, SCFAs are known to regulate mitochondrial function in the gut [40], lymphoblastoid cells [41], hepatocytes [42], beta cells [5], and adipose tissue [43]. Nonetheless, the relationship between SCFAs and cerebrovascular endothelial cells is not known, and our data revealed a novel mechanism by which SCFAs regulate mitochondrial function in HBMECs. Our studies demonstrated that SCFAs, by increasing HO-2, improve mitochondrial function during stress, such as exposure to Ang-II.
Fecal SCFA levels were shown to play an important role in reducing the body weight of high-fat diet-induced obese mice (HFD) [6]. Additionally, treatment with exogenous acetate, propionate, or butyrate has been shown to prevent weight gain in HFD mice and overweight humans [44,45]. These findings provide insights into new targeting mechanisms of SCFAs, which may be important for preventing or treating obesity-induced cerebrovascular diseases.
Our study has shed light on a new pathway by which SCFAs could affect mitochondrial function in HBMECs during stress through the regulation of HO-2. SCFAs are known to be affected by hypertension [46] and neuropathological diseases such as Alzheimer's disease [47]. Thus, examining this mechanism in vivo using a disease model known to produce gut dysbiosis-induced alteration in SCFAs levels, such as hypertension or obesity, will support a translational pipeline connecting SCFAs to cerebrovascular function.
Clinical significance: In recent years, there has been a growing body of evidence supporting the role of gut bacteria, which plays a pivotal part in the regulation of the onset and progression of cerebrovascular diseases. Benakis et al. demonstrated that gut dysbiosis occurs in several animal models of ischemic stroke. Specifically, their studies exhibit that gut microbiota can regulate neuroinflammatory responses and thereby influence brain recovery [48]. The data from this study highlights the delicate play between the brain and gut microbiome following acute brain injury. Additionally, Xiong et al. highlight the taxonomic and functional bacteria changes between patients with intraparenchymal hemorrhage compared to healthy individuals [49]. This data strongly supports the hypothesis that gut microbiota is a target of intracerebral hemorrhage-induced systemic alteration. Consequently, gut dysbiosis could have a substantial impact on the outcome of intracerebral hemorrhages and establishes the connection between gut microbiome health and cerebrovascular perfusion. Furthermore, the gut microbiota has been shown to contribute to cerebral small vessel disease [50], and the pathophysiology of cranial aneurysms by modulating inflammation [51]. It is important to note that gut microbiota (gut dysbiosis) has not only been linked to several cerebrovascular conditions such as ischemic stroke, intracerebral hemorrhage, intracranial aneurysm, and cerebral microvascular disease but also to diseases that impact the cerebrovascular physiology such as obesity and hypertension. Although the influence of gut microbiota on obesity and hypertension has been extensively studied, less is known about the effect of gut dysbiosis on obesity-induced cerebrovascular diseases. The gut microbiota communicates with the brain through its metabolites. Several studies have shown that bacterial metabolite profiles were altered in patients with various brain diseases [52]. SCFAs are gut microbiota-derived metabolites that regulate the gut-brain axis and are speculated to impact the cerebrovascular physiology following gut dysbiosis. It has been shown that SCFA are involved in neurodegenerative diseases including Alzheimer's [53], Autism [54], and Parkinson's [55]. Additionally, SCFAs show effectiveness in improving post-stroke recovery via an immunological mechanism [56]. The exact mechanism by which SCFAs affect cerebrovascular physiology is yet to be determined. In our in vitro study, we elucidate a potential mechanism by which SCFA could influence cerebrovascular physiology. We show that SCFA was able to restore the level of HO-2 and therefore rescue the cerebral mitochondrial and endothelial function. Extrapolating this data to an in vivo model of cerebrovascular disease is of great clinical significance since it could be a key step in developing novel therapeutic targets to treat central nervous diseases. Furthermore, our results provide a framework for molecular studies to better characterize the molecular mechanisms of SCFAs.