**5. miRNAs and RAS: cerebrovascular regulation and cognitive function**

### **5.1. MiR-1/206**

The miR-1/206 family has been suggested to exclusively target AT1R in the RAS; however, it has an estimate of 790 other transcripts regulating other systems [48]. MiR-1 and miR-206 are located in chromosomes 20 and 6, respectively and share homology in the seed region. An evaluation of biochemical, cardiovascular and performance indexes of aerobic exercise activity showed that some miRNAs were significantly increased. Specific correlations were found between miR-1, miR-133a and miR-206 and performance parameters, with miR-206 having the strongest positive correlation [78]. MiR-1 was also found to be decreased 1.4-fold in post-mortem cardiac tissue from acute myocardial infarction patients [79]. In contrast, elevated plasma miR-1 levels were reported to predict heart failure after acute myocardial infarction although they returned to basal levels after medication [80].

In conditions of hypoxia such as infarcts, oxygen/glucose deprivation or with ischaemia/ reperfusion intervals, miR-1 is highly expressed [79, 80]. Under less stressful and non-lifethreatening situations, miR-206 is transcribed [78], both of them targeting AT1R to decrease Ang II-mediated vasoconstriction and in doing so increasing the supply of oxygen and glucose to cells to prevent apoptosis.

MiR-1 overexpression inhibits contractility and proliferation of human vascular smooth muscle cells (VSMCs) in vitro in a negative feedback loop [81, 82]. MiR-1 is downregulated in VSMCs from spontaneous hypertensive rats and its overexpression in vivo inhibits the proliferation of VSMCs by targeting insulin-like growth factor 1 (IGF1) [83]. By contrast, miR-1 upregulation enhances angiogenic differentiation of human cardiomyocyte progenitor cells [84]. The opposite effects of miRNA in different cell types may be explained by its cell-specific expression. Indeed, even if the miRNA is expressed under physiological conditions, variations to this will depend on local gene expression in a time- and cell type-dependent manner.

Evidence of peripheral and central roles for miRNAs was seen in a transgenic mouse model of cardiac-specific overexpression where miR-1 levels were increased not only in the heart but also in the hippocampus and peripheral blood. Furthermore, the mice showed cognitive impairment by downregulation of brain-derived neurotrophic factor (BDNF), a target of miR-1 [85], providing strong evidence for a role in endocrine signalling and association between vascular disorders and cognitive impairment. Nevertheless, it is unlikely that the response depends exclusively on miR-1 and it is not known as to whether the associations are primary or secondary in nature.

Collectively, miR-1 may serve to support protective mechanisms to adapt to adverse hypoxic insults and remodel the proteome as a result. Indeed, as mentioned above, remote ischaemic conditioning showed a high correlation between ischaemia/reperfusion intervals and the levels of miR-1 in rats independent of BDNF mRNA and protein levels [69]. Hence, the miR-1/206 family is likely important in cardioprotection, prevention of stroke and consequently cognitive impairment. Already it is used in screening for myocardial infarction, monitoring and response to therapy and also has a tentative therapeutic use for increasing vasodilation and angiogenesis [86, 87].

However, a solitary miRNA or miRNA-target interaction, such as between miR-1/206 and AT1R, is unlikely to be able to explain a complete physiological response. Inherent properties between miRNA transcription, interactions between their targets, the timing of their expression and subcellular localization provide a more likely explanation. A panel of dysregulated miRNAs is likely to cause an imbalance in targets, proteins and pathways involved. Such a characteristic combination of altered miRNAs may be useful as diagnostic tools. For example, a diagnosis of cholangiocarcinoma can now be made with 100% accuracy in the presence of a 30-miRNA signature, three of them are useful for prognosis and monitoring and one of which has already entered a Phase I clinical trial as a potential treatment [88–91].

**5. miRNAs and RAS: cerebrovascular regulation and cognitive function**

1 miR-3163

example, miR-3163, is given at the bottom of the table. miRNAs in bold are described further in the text.

**Table 2.** A summary of the subgroups of miRNAs according to their functional effect in the RAS.

The miR-1/206 family has been suggested to exclusively target AT1R in the RAS; however, it has an estimate of 790 other transcripts regulating other systems [48]. MiR-1 and miR-206 are located in chromosomes 20 and 6, respectively and share homology in the seed region.

**5.1. MiR-1/206**

AGT AGTR1 AGTR2 DPP3 ENPEP LNPEP MME RNPEP

**RAS components microRNA families in common**

180 Renin-Angiotensin System - Past, Present and Future

ANPEP DPP3 ENPEP LNPEP 1 miR-670-3p

AGTR2 LNPEP MAS1 1 miR-23-3p

1 **miR-125-5p**

ENPEP LNPEP MME 17 miR-26-5p, miR-30-5p, **miR-132-3p/212-3p**, miR-

ACE2 ENPEP LNPEP 4 miR-374-5p/655-3p, miR-543, miR-4424,

ACE2 LNPEP MME 3 miR-374a-3p, miR-3194-3p, miR-5691

ACE2 DPP3 LNPEP 1 miR-329-3p/362-3p ACE2 ENPEP MME 1 miR-140-3p.1

DPP3 ENPEP 1 let-7-5p/98-5p MAS1 1 **miR-143-3p**

ACE AGTR1 DPP3 1 **miR-34-5p/449-5p** AGTR1 1 **miR-1-3p/206**

DPP3 LNPEP MME 5 miR-146-5p, miR-183-5p.1, miR-589-5p, miR-

ENPEP LNPEP 17 miR-9-3p, miR-19-3p, miR-29-3p, miR-34b-

ENPEP 13 **miR-133**, miR-142-3p.2, miR-219-5p, miR-371a-3p,

From 164 combinations of overlapping targets and miRNAs in common, only 14 are included here, 12 which if increased would favour vasoconstriction and 2 would increase vasodilation. Others tend to influence multiple RAS pathways, an

194-5p, miR-204-5p/211-5p, miR-216-5p, miR-376-3p, miR-376c-3p, miR-378-3p, miR-450b-5p, miR-518d-5p/519-5p, miR-522-3p, miR-580-3p, miR-653-5p, miR-1269, miR-3942-5p, miR-4766-3p

5p/449c-5p, miR-105-5p, miR-122-5p, miR-144-3p, miR-320, miR-323-3p, miR-323b-3p, miR-382-3p, miR-494-3p, miR-514a-5p, miR-515-5p/519e-5p, miR-642a-5p, miR-3146, miR-5579-3p

miR-409-5p, miR-451, miR-496.1, miR-508-3p, miR-526b-5p, miR-877-5p, miR-1185-5p, miR-5094

miR-1306-5p

876-5p, miR-2355-5p

AGTR2 DPP3 LNPEP MME 1 miR-17-5p/20-5p/93-5p/106-5p/519-3p ACE2 ENPEP LNPEP MME 3 miR-9-5p, miR-200-3p/429, miR-942-5p

ACE ACE2 ANPEP ENPEP

LNPEP

### **5.2. MiR-143**

The Mas receptor (MasR) has the lowest number of associated miRNAs, implying steady and tightly regulated homeostatic expression, although other post-transcriptional modifications are also likely to be involved in its regulation. In addition, miR-143 is exclusive to MasR in the RAS and interestingly, it has been found to be dysregulated in vascular disorders [92]. MiR-143 is enriched in cardiac stem cells before becoming localized to smooth muscle cells, including neural vascular smooth muscle cells (VSMC) in mice and its expression was found to be dependent on heartbeat rate in zebrafish [93, 94]. In human peripheral blood mononuclear cells, miR-143 was upregulated in patients with essential hypertension and decreased in aortic aneurysms [95, 96]. Previous studies have focused on other targets of miR-143 in hypertension, yet the potential effect of miR-143 via the MasR remains elusive. Due to the small number of miRNAs attributed to the regulation of MasR, fluctuations in just one of them might have a significant effect on MasR protein levels.

### **5.3. MiR-132/212**

Ang II regulated the miR-132/212 family in hypertensive rats and humans [97, 98] and this family has been attributed with both cardiovascular and brain-specific properties [99–103]. MiR-132/212 was initially thought to directly target AT1R with experimental studies demonstrating a prevalent effect in the RAS, but new advances and criteria in miRNAs have shown that the effect was due to various downstream second messengers of AT1R activation. miRNA-132/212 has multiple targets including Ang II and endothelin-1 (ET-1) signalling [99]. Thus, miRNA-132/212 might be relevant in hypoxic conditions to control the vasoconstrictor effects of Ang II and ET-1. Indeed, transplantation of pericyte progenitor cells from human adult vena safena (Bristol pericytes) induced pro-angiogenic activity in endothelial cells, mediated by pericyte-produced miR-132 in response to hypoxia and taken up by endothelial cells passing through exosomes [104–106].

MiR-132 expression is also regulated by CREB [107, 108], enhances the frequency and amplitude of excitatory potentials in neurons and increases dendritic length and arborization by targeting the brain-enriched GTPase-activating protein p250GAP [109, 110]. MiR-132 triggered marked increases in dendritic spine density, while either underexpression or overexpression of miR-132 caused cognitive impairment in supra-physiological conditions [100, 111]. Similarly, BDNF is regulated by CREB and a negative feedback interaction between the previously described miRNA-1/206 and miRNA-132/212 regulates BDNF expression in the brain [112]. Notably, miRNA-132/212 is also involved in the brain-immune axis and miR-132 mediates an anti-inflammatory effect by targeting acetylcholinesterase, thus increasing acetylcholine that reduces cytokine production [113, 114]. Furthermore, projections from basal forebrain neurons to cortical microvessels (nervi vasorum) and astrocytes containing primarily acetylcholine and nitric oxide synthase (NOS) have contributed to increased cerebral blood flow [77].

### **5.4. MiR-29**

Another miRNA family dysregulated in cerebrovascular disorders and regulated by Ang II is miR-29 [74, 98]. The miR-29 family is linked to cardiac and vascular ageing and counteracts fibrosis by regulating extracellular matrix metallopeptidases [115]. Ang II increased miR-29b in cardiac fibroblasts with no effect in myocytes [116]. In the renal cortex of spontaneously hypertensive rats and in renal tubular epithelial cells, Ang II decreased the expression of miR-29b [117]. Notably, ET-1 decreased miR-29a expression in cardiac myocytes in vitro [118]. MiR-29b is increased in rat brain after focal ischaemia in vivo and in primary neurons exposed to oxygen/glucose deprivation in vitro [119]. Treatment of rats with peroxisome proliferator-activated receptor gamma (PPARγ) agonists protected against ischaemia-reperfusion injury by decreasing miR-29a and miR-29c levels; correspondingly, apoptosis was induced by overexpressing miR-29 [120]. However, mouse models of middle cerebral artery occlusion have inconsistently demonstrated increased and reduced miR-29 levels [119, 121–123]. These conflicting findings have a number of possible explanations including animal age and species, as well as techniques and biosamples used, or other factors discussed below.

Despite the inconsistent evidence, a meta-analysis of microRNAs induced by aerobic exercise in humans evaluated left ventricle hypertrophy and proposed miR-29 family to be antihypertrophic and miR-34 family to be prohypertrophic [124]. MiR-34 was increased in patients with cardiovascular disorders in response to stress [125], which promotes apoptosis and cardiac autophagy [102]. By contrast, myocardial hypertrophy induced by Ang II/AT1R activation in rats is antagonized by miR-34 and its inhibition stimulated Ang II signalling via atrial natriuretic peptide [126]. AT1R activation increased intracellular calcium levels producing vasoconstriction in vascular smooth muscle cells. In addition, in endothelial cells, elevation of intracellular calcium levels contributes to the inhibition of nitric oxide production by atrial natriuretic peptide [127].

### **5.5. MiR-34**

**5.2. MiR-143**

182 Renin-Angiotensin System - Past, Present and Future

**5.3. MiR-132/212**

**5.4. MiR-29**

The Mas receptor (MasR) has the lowest number of associated miRNAs, implying steady and tightly regulated homeostatic expression, although other post-transcriptional modifications are also likely to be involved in its regulation. In addition, miR-143 is exclusive to MasR in the RAS and interestingly, it has been found to be dysregulated in vascular disorders [92]. MiR-143 is enriched in cardiac stem cells before becoming localized to smooth muscle cells, including neural vascular smooth muscle cells (VSMC) in mice and its expression was found to be dependent on heartbeat rate in zebrafish [93, 94]. In human peripheral blood mononuclear cells, miR-143 was upregulated in patients with essential hypertension and decreased in aortic aneurysms [95, 96]. Previous studies have focused on other targets of miR-143 in hypertension, yet the potential effect of miR-143 via the MasR remains elusive. Due to the small number of miRNAs attributed to the regulation of MasR, fluctuations in just one of

Ang II regulated the miR-132/212 family in hypertensive rats and humans [97, 98] and this family has been attributed with both cardiovascular and brain-specific properties [99–103]. MiR-132/212 was initially thought to directly target AT1R with experimental studies demonstrating a prevalent effect in the RAS, but new advances and criteria in miRNAs have shown that the effect was due to various downstream second messengers of AT1R activation. miRNA-132/212 has multiple targets including Ang II and endothelin-1 (ET-1) signalling [99]. Thus, miRNA-132/212 might be relevant in hypoxic conditions to control the vasoconstrictor effects of Ang II and ET-1. Indeed, transplantation of pericyte progenitor cells from human adult vena safena (Bristol pericytes) induced pro-angiogenic activity in endothelial cells, mediated by pericyte-produced miR-132 in response to hypoxia and taken up by endothelial

MiR-132 expression is also regulated by CREB [107, 108], enhances the frequency and amplitude of excitatory potentials in neurons and increases dendritic length and arborization by targeting the brain-enriched GTPase-activating protein p250GAP [109, 110]. MiR-132 triggered marked increases in dendritic spine density, while either underexpression or overexpression of miR-132 caused cognitive impairment in supra-physiological conditions [100, 111]. Similarly, BDNF is regulated by CREB and a negative feedback interaction between the previously described miRNA-1/206 and miRNA-132/212 regulates BDNF expression in the brain [112]. Notably, miRNA-132/212 is also involved in the brain-immune axis and miR-132 mediates an anti-inflammatory effect by targeting acetylcholinesterase, thus increasing acetylcholine that reduces cytokine production [113, 114]. Furthermore, projections from basal forebrain neurons to cortical microvessels (nervi vasorum) and astrocytes containing primarily acetylcholine and

nitric oxide synthase (NOS) have contributed to increased cerebral blood flow [77].

Another miRNA family dysregulated in cerebrovascular disorders and regulated by Ang II is miR-29 [74, 98]. The miR-29 family is linked to cardiac and vascular ageing and

them might have a significant effect on MasR protein levels.

cells passing through exosomes [104–106].

MiR-34 is involved in cardiac and endothelial senescence, characterized by decreased production of the vasodilator nitric oxide by endothelial nitric oxide synthase, inflammation and resultant endothelial dysfunction [128]. MiR-34 promotes endothelial senescence by downregulating the histone deacetylase sirtuin-1 [129] and regulates cardiac contractile function during ageing and after acute myocardial infarction, as a result of inducing DNA damage and telomere attrition [130]. Transplantation of bone marrow-derived mononuclear cells from patients with cardiovascular disease induced cell death, while inhibition of the elevated levels of miR-34a ex vivo improved the functional benefit of transplanted bone marrow-derived mononuclear cells in mice after acute myocardial infarction in vivo [131]. Inhibition of miR-34 also attenuated ischaemia-induced cardiac remodelling, atrial enlargement and improved systolic function [125, 132].

By contrast, miR-34 promoted differentiation of mouse embryonic neural stem cells to postmitotic neurons by targeting sirtuin-1 [133]. Along with miR-132/212, miR-34 was upregulated in human epilepsy screenings and pilocarpine-induced status epilepticus in rats [134–139], suggesting neuronal activity-based regulation. MiR-34 expression in the amygdala is also linked to repression of stress-induced anxiety [140], modulates ageing and neurodegeneration in Drosophila [141] and is associated with cognitive impairment [142].

The miRNA families described are functionally relevant in the development of cardiovascular and cerebrovascular disorders, some of which appear to link cerebral ischaemia, endothelial dysfunction and cognitive impairment. Current therapy for cerebral ischaemia is limited to the use of recombinant tissue-plasminogen activator (tPA). Endogenous tPA is primarily expressed in endothelial cells and interactions between tPA and low-density lipoprotein receptor-related protein (LRP) are important for the hippocampal activity-dependent strengthening of synapses known as long-term potentiation (LTP) [143]. AT1R activation causes increased expression of tPA inhibitor (tPA-I), which binds to LRP and blocks its interaction with other ligands, including apolipoprotein E and alpha 2-macroglobulin [144]. Furthermore, tPA-I limits the maturation of proBDNF to BDNF and impedes protein synthesis-dependent late-phase LTP and hippocampal plasticity, mechanisms for learning and memory [145]. Chronic administration of tPA improved cognition in a APPswe/PS1 transgenic mice [146]. MiR-34 has two different binding sites at the 3′UTR of tPA-I, one of which has the highest probability of binding amongst the 108 miRNAs for this transcript. LRP1 is subject to regulation by 22 miRNAs, including miR-125 with one binding site and miR-212 with two binding sites [48].

There has been a recent consensus view on the roles of microRNAs, platelet and endothelial dysfunction in vascular disease and inflammation [147]. MiR-132/212 and miR-29 families target some proteins involved in endothelial dysfunction, such as the actin-related protein 2/3 complex, platelet-derived growth factor and aquaporin 4 [48]; the latter two are particularly relevant in the maintenance of blood-brain barrier (BBB) integrity [148, 149]. Factors involved in BBB disruption include chronic hypertension, ischaemia, trauma, infections and inflammation. Throughout the life course, these factors are likely to cause epigenetic modifications including miRNA fluctuations, leading to reduced protein translation and degradation of mRNA transcripts necessary for BBB integrity. BBB disruption is relevant in understanding the spectrum of clinical manifestations resulting from cerebrovascular disorders.
