University of Groningen

Brain and retinal macro- and microvasculatureLi, Youhai

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CHAPTER 6

General discussion

CHAPTER 6

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Summary and general discussion

The vasculature of the central nervous system (CNS) is divided into the macrovessels

and microvessels, both of which are composed of two principle cell types: the

endothelial cells (ECs) lining the inner walls of the vessels, and mural cells (pericytes

or smooth muscle cells) enveloping the abluminal surface of the endothelial tube. The

macrovessels, most notably the small arteries and arterioles control cerebral blood flow

(CBF) and propel the oxygenated blood forward into downstream microvascular

networks, whereas the microvessels are responsible for modulating the exchange of

gases, nutrients, hormones, ions and metabolites between the circulating blood and

parenchyma.

Dysfunction of ipsilateral middle cerebral artery after acute ischemic stroke

Autoregulation of CBF is the ability of the brain vasculature to maintain constant blood

flow despite changes in blood pressure (1). The normal vasomotor activity of the middle

cerebral artery (MCA) is a critical contributor to the CBF autoregulation. In chapter 2,

we designed experiments to investigate the alterations of MCAs after 24 hours of

ischemia/reperfusion (I/R) injury. After acute ischemic stroke, the diameter of the

ipsilateral middle cerebral artery (MCA) was significantly enlarged compared to that

of contralateral MCA (Fig. 1) indicating that the ischemic MCA became dysfunctional

including loss of autoregulation and failure to appropriately control blood flow to the

ischemic area. This may lead to a worsening of the outcome of ischemic stroke as it can

aggravate brain edema or facilitate hemorrhagic transformation (2).

Previous studies reported that in cerebral ischemic stroke the myogenic activity of

ischemic MCA was diminished (3, 4). It was suggested that the loss of myogenic tone

of ischemic MCA might be associated with dysfunction of smooth muscle cells (SMCs).

In chapter 2, we observed that the contractile ability of the SMCs of MCAs upon

U46619 (a stable thromboxane A2 (TP) receptor agonist) administration was not

affected by I/R injury (Fig. 1; Li Y et al, unpublished data). To uncover the underlying

molecular mechanisms of I/R induced MCA dysfunction, we further tested the

functions of endothelial cells (ECs) and its signaling interactions with SMCs upon

treatment with specific agonists and/or antagonists.

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Figure 1. Abnormal dilatation of the ipsilateral middle cerebral artery (MCA) following

an acute ischemic stroke in rats. A) Representative images of a rat brain following 24 hours

ischemia/reperfusion (I/R) injury. B) The diameter of ipsilateral MCAs is significantly enlarged

compared to that of contralateral MCAs (upper panel), whereas the contractile abilities of

MCAs from both sides were not different (lower panel). The contractile force of MCAs was

calculated after administration of the thromboxane A2 (TP) receptor agonist, U46619 (9,11-

Dideoxy-9a,11a-methanoepoxy prostaglandin F2a, 0.3 μM). *Significantly different from

contralateral MCAs.

Upregulation of B2 receptors in the endothelial cells of the ischemic MCAs

In chapter 2, we describe that bradykinin (BK), a potent endothelium-dependent

vasodilator, produced a significantly higher relaxation in ipsilateral MCAs compared

to either contralateral MCAs or MCAs from healthy rats. Previous studies demonstrated

that BK mediates its biological effects via the activation of two receptor subtypes,

namely the B1 and B2 receptor, both belonging to the seven-transmembrane G-protein

coupled receptor superfamily (5). We found that in the ischemic MCAs preincubation

with the B1 receptor antagonist lys-(des-arg , leu )-BK had no effect on the BK-

induced relaxation, whereas the B2 receptor antagonist icatibant completely suppressed

the BK-induced relaxation. Furthermore, we showed that B2 mRNA expression was

significantly increased in ipsilateral MCAs compared to contralateral MCAs. BK-

induced relaxation was completely abolished in endothelium-denuded MCAs. The B2

receptor protein was detected in the ECs of the ipsilateral MCAs using

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immunofluorescence staining. These results suggest that activation of B2 receptor

accounts for the BK-induced relaxation, and in the ipsilateral MCAs B2 receptor was

upregulated by the I/R injury.

Signaling pathways of the BK-induced relaxation in the ischemic MCAs

The activation of B2 receptors in ECs will increase intracellular calcium [Ca2+]i, which

can trigger downstream pathways (6). Nitric oxide (NO) is a pivotal vasodilator for the

endothelium-dependent relaxation by stimulating NO-sensitive guanylyl cyclase in

SMCs (7). In mammals, NO can be generated by three different isoforms of the enzyme

nitric oxide synthases (NOS): the neuronal isoform (nNOS), the inducible isoform

(iNOS) and the endothelial NOS (eNOS) (8). We show in chapter 2, that the NOS

inhibitor nitroarginine (L-NNA) or a soluble guanylyl cyclase (sGC) inhibitor ODQ

(1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one) almost abolished the sustained

relaxant responses to BK while uncovering a transient response in these segments. This

observation suggests the involvement of additional signaling pathway, e.g.,

endothelium-dependent hyperpolarization (EDH). Several Ca2+-dependent K+ channels

(Kca) have been proposed to regulate the EDH process, such as intermediate

conductance Kca channel (IKca), small conductance Kca channel (SKca) and big

conductance KCa channel (BKca) (9-12). Our results showed that the transient

relaxation upon BK occurring in MCAs with the NO-sGC pathway inhibited was

completely removed by charybdotoxin (a blocker of BKca and IKca), but not apamin

(a selective blocker of SKca) or iberiotoxin (a selective blocker of BKca), suggesting

that IKca channel activation accounts for the BK-induced EDH. Taken together, in

chapter 2 we demonstrate that upregulation of B2 receptor expression within

endothelium of ipsilateral MCAs is responsible for the BK-induced significant

vasodilatation, which is mediated via two signaling pathways between ECs and SMCs

(Fig. 2). Our findings demonstrate for the first time that the function of endothelial cells

in ischemic MCAs is altered by I/R injury. The main effector appears to be a significant

upregulation of the underlying B2 receptor expression.

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Figure 2. Schematic paragraph showing the signaling pathways of the BK-induced

relaxation in the middle cerebral artery (MCA) after acute ischemic stroke. BK,

bradykinin; eNOS, endothelial nitric oxide synthase; IKca, intermediate conductance Kca

channel; EC, endothelial cell of the ischemic MCA; SMC, smooth muscle cell of the ischemic

MCA.

Newly established protocol for isolating brain and retinal microvessels from

healthy and diabetic rats

The molecular exchange between circulating blood and CNS parenchyma takes place

in the microvascular networks, which mainly consist of two interacting cell types:

endothelial cells (ECs) and pericytes (PCs). In order to explore the cellular and

molecular properties of retinal and brain microvasculature, in chapter 3, we first

developed a new mechanical method for isolation of retinal microvessels (RMVs) and

brain microvessels (BMVs) from the same rats. Morphometric analysis showed that the

density of PCs in RMVs was significantly higher than in BMVs, in terms of the mean

distance of neighboring PCs (45 μm in RMVs versus 61 μm in BMVs) and the

endothelial cell/pericyte ratio (1.8 in RMVs versus 2.7 in BMVs). This is consistent

with previous reports, in which the pericyte coverage in BMVs (either rat or monkey)

was significant less as compared to RMVs (13, 14). In addition, our gene expression

data show significant accumulation of endothelial cell- and pericyte-specific markers

in the RMVs and BMVs isolated from both healthy and diabetic rats. These results

demonstrate that our new established protocol can yield high amounts and highly

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purified RMVs and BMVs from both healthy and diabetic rats, and the structural

integrity of these isolated microvessels is well preserved

Different susceptibility to diabetes

In mammals, brain and retinal microvasculature form two comparable blood-neuronal

barriers, namely blood-brain barrier (BBB) and inner blood-retinal barrier (iBRB) (15,

16) for maintaining a microenvironment that allows neurons to function properly. The

molecular movements in CNS between blood and parenchyma are tightly regulated by

the ECs barriers that are characterized by the presence of extensive intercellular

junctions, absence of fenestrations and an extremely low rate of transcytosis (17).

Pericytes partially embrace the abluminal side of ECs (range from 10% to 50%

depending on the supplied tissue) and send finger-like projections inserted into

endothelial invaginations with N-cadherin–mediated peg-and-socket junctions (18).

Many studies have demonstrated that pericyte plays a crucial role in the development

and maintenance of BBB and iBRB (19, 20). In diabetes, reduction of pericyte coverage

on the retinal microvasculature has been proposed as a key event that contributes to

microvascular dysfunction including the breakdown of iBRB and the formation of

acellular capillaries in diabetic retinopathy (21-23). However, in the brain

microvasculature, the pericyte coverage is not changed by diabetes (24). This

interesting phenomenon produced two questions: why BMVs that have less pericyte

coverage do not show microvessel leakage, in other words, how BMVs are able to keep

PCs and ECs stabilization in chronic hyperglycemia of diabetes.

Molecular interactions between endothelial cells and pericytes of microvessels

During the development of the microvessels, sprouting ECs synthesize and secrete

platelet derived growth factor beta (PDGFb), which binds with high affinity to the

pericyte-specific receptor PDGFRb, resulting in the proliferation and recruitment of

PCs (25). There are two basic purposes/functions for PCs embracing on the abluminal

side of ECs: (i) stabilizing newly formed endothelial tubules (26) and (ii) contributing

to the formation/maintenance of endothelial barriers (e.g., BBB and iBRB) (19). Tight

regulation and close coordination between ECs and PCs are necessary to form a

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network of mature microvessels. At a molecular level, the pericyte coverage on

microvessels is regulated by the crosstalk between ECs and PCs. Several signaling

pathways have been demonstrated to be pivotal in the ECs/PCs interactions, such as the

PDGFb/PDGFRb signaling pathway (as mentioned above), the angiopoietin/Tie2

signaling pathway and the TGFβ signaling pathway (27). Activation of Tie2 receptors

(in ECs) by angiopoietin 1 (Ang1, from PCs) promotes vessel stabilization and

maturation (28), whereas angiopoietin 2 (Ang2, in ECs) binding to Tie2 triggers cell

migration, proliferation and vessel leakage (29, 30). Similarly, the biological effects of

TGFβ also depend on its receptors: activation of activin receptor-like kinase 5 (Alk5,

also known as TGFβ receptor-1) will promote vessel stabilization while Alk1 activation

triggers cell migration, proliferation and vessel destabilization (28). In chapter 4, using

transcriptome analysis, we found that expression of Ang1 in BMVs was significantly

higher than in RMVs, while Tie2 and Ang2 expression were comprable between BMVs

and RMVs. These results indicate that the microvessel stablity and maturation remains

more secure in BMVs than in RMVs.

In addition to the signaling crosstalk, ECs also directly contact with PCs in areas

lacking the basement membrane, through abundant ECs-PCs gap junctions (GJs) made

up by different connexins (e.g., Cx37 and Cx43) (31). Previous studies demonstrated

that reduction of GJs expression contributed to compromised vascular homeostasis (32),

impaired mural cell differentiation (33) and endothelial barrier damage (34, 35). In

chapter 4, we observed that the expression levels of Cx37, Cx40 and Cx43 were

significantly higher in BMVs than in RMVs. Therefore, one may conclude that higher

expression of Ang1 and connexins genes in BMVs in turn enable BMVs to have more

intravascular stabilization than in RMVs.

Effects of chronic hyperglycemia on retinal and brain microvasculature

In brain and retina, blood glucose is transported rapidly through the ECs by the glucose

transporter 1 (Glut-1) (36). After cellular uptake, glucose in ECs is mainly split

into pyruvate for adenosine triphosphate (ATP) production via the aerobic glycolysis

pathway (Fig. 3, 4) (37). In the past decades, multiple molecular mechanisms have been

proposed to explain the pathogenesis of diabetic microvascular complications,

including hyperglycemia-induced reactive oxidative species (ROS) overproduction,

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accumulation of advanced glycation end products (AGEs), activation of protein kinase

C (PKC) and NF-κB pathway activation (Fig.3) (38-41). Most of these theories are

based on studies using hyperglycemia-susceptible organs (e.g., retina), but they never

elucidate why the closely related brain microvasculature is not or less susceptible to

diabetes.

In chapter 5 we found in diabetes, that gene expression patterns of BMVs and RMVs

were distinct. Depending on the functional properties, these altered genes are divided

into four major categories as described below.

i) Genes related to ROS production. Stanniocalcin-1 (Stc-1) and Bhlhe40 are two

enzymes that can suppress the ROS generation (42-46). NAD(P)H dehydrogenase 1

(Nqo1) and Glutathione S-transferase P (Gstp1) are pivotal antioxidant enzymes in

response to DNA damage by ROS injury (47-49). Under diabetic conditions, Stc-1 and

Bhlhe40 genes were significantly upregulated in BMVs, while Nqo1 and Gstp1 genes

were significantly downregulated in RMVs, suggesting that in BMVs the oxidative

stress induced by diabetes might be counterbalanced by antioxidants although more

studies are required to elucidate this relation.

ii) Genes related to methylglyoxal production. Methylglyoxal (MG), a major precursor

of advanced glycation end products (AGEs), is derived primarily from

dihydroxyacetone phosphate (DHAP) (50, 51). Under diabetic conditions, expression

of glycerol-3-phosphate dehydrogenase 1 (Gpd1) was significantly upregulated in

BMVs whereas it was not changed in RMVs. Previous studies showed that Gpd1 is an

enzyme that converts DHAP into glycerol-3-phosphate (G3P) with a decrease in the

NADH/NAD+ ratio (52). This process can reduce cellular concentration of DHAP and

prevent the spontaneous conversion of DHAP into MG (53). The overexpression of

Gpd1 in BMVs might reduce MG formation in the brain microvasculature, thereby

diminishes the diabetic insults.

iii) Genes related to inflammation. Neuron-derived orphan receptor 1 (Nr4a3) and

TSC22 domain family protein 3 (Tsc22d3) have anti-apoptotic effects through

prevention of NF-κB activation (54-56), while TNF receptor superfamily member 21

(Tnfrsf21, so-called DR6) can activate the NF-κB pathway and trigger cell apoptosis

(57). Under diabetic conditions, we observed that in BMVs, Nr4a3 and TSC22 genes

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were significantly upregulated, meanwhile Tnfrsf21gene was significantly

downregulated. All these genes were not changed in RMVs, suggesting that in diabetes,

the anti-inflammatory capacity of BMVs seems to be enhanced by inhibition of the NF-

κB pathway.

iv) Genes related to protein translation. Aminoacyl-tRNA synthetases (aaRSs)

catalyze the ligation of amino acids to their cognate tRNAs, thereby playing a key role

in protein synthesis (58). In addition to the aminoacylation function, many studies have

shown that aaRSs have multiple non-canonical functions, e.g., regulation of glucose

metabolism and inflammation (59, 60). Under diabetic conditions, we observed that

expression of Nars, Gars, Mars, Iars and Yars (5 components of aaRSs) in RMVs were

significantly upregulated. Previous studies have shown that oxidative stress can cause

damage to aaRSs functions, followed by amino acid mistranslation and protein

misfolding (61, 62). We thus speculate that in RMVs, hyperglycemia-induced

superoxide causes upregulation of aaRSs genes, which in turn affects the glucose

metabolism, inflammation and reliability of protein translation in RMVs (Fig. 3).

Taken together, we demonstrated that in diabetes, BMVs have multiple defense

mechanisms including reduction of ROS production, reduction of glycolytic

intermediates and enlarged anti-inflammatory capacity, against the detrimental effects

of diabetes (Fig. 4). In contrast, these protective systems were not activated or even

suppressed in RMVs (Fig. 3). Based on these findings, it is not surprising to observe

that the brain microvasculature is less susceptible to diabetes as compared to the retinal

microvasculature.

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Figure 3. Schematic representation of the molecular mechanisms of retinal microvessels

in the hyperglycemia of diabetes.

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Figure 4. Schematic representation of the defense mechanisms of brain microvessels in

the hyperglycemia of diabetes. Blood glucose is transported to the brain microvascular

endothelial cells by the glucose transporter 1

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Conclusions

With regard to macrovesssels, our studies demonstrate that after the acute ischemic

stroke, the B2 receptor in the ECs of ischemic MCA was overexpressed, and activation

of B2 receptor lead to MCA dilatation via two signaling pathways between ECs and

underling SMCs: 1) the B2-eNOS-sGC signaling pathway, and 2) B2-IKca-EDH

signaling pathway. Upregulation of B2 receptor likely contributes to the abnormal

enlargement of ischemic MCA. With regard to microvessels, we developed a new

method for both RMVs and BMVs isolation from individual rats. The morphological

analysis demonstrated that in rat, the pericyte density in RMVs was higher as compared

to BMVs, in term of EC/PC ratio and PC-PC distance. In physiological conditions, we

have shown that the expression levels of Cx37, Cx40, Cx43 and Ang1 were

significantly higher in BMVs as compared to RMVs. Under diabetic conditions, we

have shown that several compensatory mechanisms appeared in BMVs, including

reduction of glycolytic intermediates, reduction of ROS production and enlarged anti-

inflammatory capacity, whereas these defense systems were not activated or even

suppressed in RMVs. These findings help to understand why retinal and brain

microvasculature show different susceptibilities to diabetes, which may contribute to

new approaches in drug development for the treatment of vascular complications in

diabetes.

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Future perspectives

Part I: Utilization of the new protocol for microvessel isolation

In this thesis, we have described a new protocol for isolating rat RMVs and BMVs,

which were used to identify the diabetes changed genes. In the next step we will use

our protocol to isolate the RMVs and BMVs from acute brain ischemic rats and to

assess the gene expression alterations of those microvascular beds in the early stage of

ischemic stroke. Our preliminary studies showed that this protocol can be used with a

little modification for isolating the BMVs from ob/ob mice (Li Y et al, unpublished

data). With the help of this modified protocol, we will be able to explore the molecular

profiles of mice brain microvascular beds in front of diabetic or ischemic stroke insult.

In the future we also can modified our protocol to isolate human RMVs and BMVs in

both healthy and pathological conditions (e.g. diabetes and ischemic stroke), which

might help to uncover the molecular mechanisms of these diseases.

Figure 5. The microscopic images showing the high amount and high purity of brain

microvessels isolated from one ob/ob mice. Images are stained with haemotoxylin and eosin

(H&E).

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Part II: Molecular functions of the diabetic changed genes in RMVs and BMVs

Our animal studies demonstrated that in diabetes the gene expression patterns of retinal

and brain microvasculature were distinct. The functional properties of these identified

genes were descripted according to the previous observations, such as Nr4a3 preventing

the activation of NF-κB pathway (55), Gpd-1 preventing the formation of MG (53).

Based on the previous literature, we speculated that in diabetes, the BMVs expressed

multiple complementary mechanisms to diminish the toxic effects of diabetes, whereas

these processes are not activated or suppressed in the RMVs. However, because most

of those reports were performed on non-vascular cells, the functional role of those genes

in vascular endothelial cells is at present unknown. Besides, the molecular mechanisms

of how these genes were transcriptionally changed are not clear. Therefore, to gain more

insight in the understanding of the transcriptional alterations of retinal and brain

microvessels in diabetes, more studies are required. Below I give an example for

uncover the functional role of diabetic changed gene.

Nr4a3, also known as NOR-1, is a ligand-independent transcription factor. Our animal

studies showed that the expression level of Nr4a3 mRNA in BMVs was significantly

increased by diabetes. Human umbilical vein endothelial cells (HUVEC) are widely

used to study the molecular processes of vasculature under high glucose conditions. In

chapter 5 of this thesis, in vitro studies on HUVECs showed that Nr4a3 mRNA

expression was significantly upregulated by high glucose treatment. To further study

the role of Nr4a3 upregulation, gene silencing to knock down Nr4a3 in HUVECs can

be used. Short interfering RNA (siRNA), a tool for inducing short-term silencing of

genes, can assist in elucidating the response of endothelial cells to high glucose in the

low expression of Nr4a3. Preliminary studies showed that Nr4a3 gene silencing using

siRNA can be accomplished with more than 70% reduction in HUVECs (Y Li et al,

unpublished data). It has been reported that Nr4a3 upregulation in vascular smooth

muscle cells can inhibit the activation of NF-κB pathway (55). The potential anti-

inflammatory function of Nr4a3 can be studied, for example, by determining the

expression of adhesion molecules (e.g. E-selectin), chemokines (e.g. interleukin-8) in

HUVECs under high glucose conditions, where Nr4a3 has been knocked down.

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