Circulating Inflammatory MRNA (KLF4 And MMP9) Associated With Occurrence And Development Of Acute Hypertensive Stroke

BackgroundStroke is one of the major killers and the leading cause of disability worldwide during the past decade. In China, the incidence of annual stroke mortality is nearly 1.6 million, which has exceeded heart disease to become the major cause of death. Ischemic stroke (IS) is the most common type of stroke in China, while hemorrhagic stroke accounts for 20% to 50%. It’s widely accepted that hypertension is the primary and independent risk factor for overall stroke. Patients with hypertension are particularly susceptible to hemorrhagic and ischemic stroke, for hypertension increases the risk of stroke 7 folds. However, only a fraction of individuals with hypertension develop stroke, indicating other potential mechanisms might participate in the development of stroke.Heredity, suggested by evidence from twin-based and family-based studies, might contribute to a susceptibility to stroke. It has been documented that genetics have a role in up to 50%. During the last decade, numerous meta-analysis of susceptible gene studies and genome wide association studies (GWAS) have been performed. The genetic association studies have showed that a series of genomic regions and variants increase the risk of stroke. Additionally, since message RNA (mRNA) can convey genetic information, it has also been studied in the genomic research. On the other hand, the expression of mRNA is influenced by genetic, epigenetic and environmental factors, so the blood mRNAs can reflect the integration of above-mentioned factors. Given that stroke is a kind of multifactorial disease, we hypothesize that the abundance of specific mRNA molecules may help to distinguish hypertensive individuals at risk of IS or ICH.Patients who suffer from hypertension have a high risk of stroke, but about 30% predispose to IS or ICH. Based on previous data, we suspect that different gene-environment interactive influences may determine the propensity for various stroke subtypes. Genetic factors that predispose to IS include inflammatory, coagulation/hemostasis, endothelial function, and renin-angiotensin-aldosterone, while to intracerebral hemorrhage (ICH) include intrinsic vascular wall integrity and malformations. Recently, it’s generally recognized that IS is caused by carotid atherosclerosis. So whether hypertensive patients with carotid vulnerable plaques are prone to develop IS. This question remains to be answered. Similarly, atherosclerosis is also associated with the ICH patients with longstanding hypertension since hypertension accelerates atherosclerosis. In our previous experiments, the significant differentially expressed miR-29a-3p is observed in ICH cases with hypertension by miRNA microarray screening. Using TargetScan (http://www.targetscan.org/), Pictar (http://www.pictar.org/) and MiRanda (http://www.microrna.org/mictorna/home.do) databases, we found all three software programs identified 103 target genes of miR-29a-3p. Among the 103 genes, considering that KLF4 is related to atherosclerosis and COL1A2 is correlated with vascular wall integrity, we selected KLF4 and COL1A2 as potential target genes for further exploration. We supposed that KLF4 and COL1A2 probably explain why a portion of hypertensive patients develop ICH. However, there is no statistical significance among the altered expression of circulating miRNAs in patients with hypertensive IS compared with hypertensive controls. So we additionally investigate whether atherosclerotic genes (sCD40L, MMP9) is associated with hypertensive IS.KLF4KLF4, as a critical regulator in the process of atherosclerosis, plays an important role in macrophage activation, vascular smooth muscle cell phenotypic modulation and endothelial dysfunction. Previous studies showed that KLF4 can promote inflammation response, while sometimes can inhibit the inflammatory response. For example, over expression of KLF4 in the vascular endothelial cells can induce expression of a series of anti-thrombotic and anti-inflammatory factors, like endothelial nitric oxide synthase enzyme (eNOS) and thrombomodulin. The two factors take part in the anti-inflammatory and anti-thrombotic process. KLF4 can also promote inflammation response. KLF4 can be produced while the macrophages are activated by inflammatory factors, such as IFN-γ、LPS、TNF-α and so on. On the contrary, KLF4 can be down regulated while macrophages are inhibited by inflammatory factors like TGF-B1.Finally, KLF4 can modulate the phenotype of smooth muscle cells. The upregulation of KLF4 inhibits the expression of marker protein of smooth muscle cells when the vessels are injured. That promotes vascular smooth muscle cells change from contractive type to secretory type, resulting in exacerbating atherosclerosis. In KLF4 knockout mice, researchers can observed a distinguish reduction in lesion size and an increase in fibrous cap thickness, which indicates increases in several indices of plaque stability.COL1A2This gene encodes collagen alpha-2(I) chain for type I collagen. Collagen type I and collagen type III accounts for about 80% of total arterial collagen. These two types of collagen alter the stabilization of the collagen fibril, influence integrity of the artery wall and reduce tissue rigidity and elasticity. Previous study revealed that collagen type I plays a more important role in the maintenance of vascular integrity. Miss ratio of the type Ⅲ/type Ⅰ collagen, like higher proportion of type I collagen, reduces vessel extensibility, which may result in vessel rupture.MMP9MMP9, belongs to the zinc-metalloproteinases family, has an effect on the degradation of the extracellular matrix. MMP9 is secreted by macrophages, endothelial cells and smooth muscle cells in response to inflammatory stimuli. MMP9 can increase the fragility of plaque by destroying the surface of the fibrous cap of plaques. In carotid atherosclerotic plaques, the more macrophages there were, the higher MMP-9 activities were. Another study showed that the levels of MMP9 in the plasma were elevated in patients suffering from cerebral embolization, which suggests that plasma MMP9 is associated with atherosclerotic plaque instability.sCD40LCD40L can be secreted by activated T cells, platelets and vascular smooth muscle cells, endothelial cells, macrophages. CD40L involves in early development of the atherosclerosis process and participates in the rupture of plaque. mCD40L and sCD40L are two main forms of CD40L. These two forms have similar functions. sCD40L, an active form of CD40L, is released from activated platelets. CD40L can induce inflammatory cells to secrete chemotactic factor, cytokines, adhesion molecules, such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E selection element, interleukin-8 and tumor necrosis factor, which participate in the atherosclerotic process. In addition, CD40L can promote macrophages and endothelial cells to produce matrix metalloproteinases, which degrade the matrix components and make the fibrous cap thinner, leading to plaque rupture.In this study, we testified the hypothesis that carotid plaque instability can predispose an individual with hypertension to IS or ICH. Then from an atherosclerosis standpoint, we aimed to study whether mRNA of circulating atherosclerotic genes and COL1A2 were associated with stroke and even stroke phenotypes in hypertensive patients. Furthermore, these results were crucial to the understanding of the etiology of hypertensive stroke and provided insights into potential mechanisms underlying the increased predisposition of some individuals to IS or ICH. Last but not the least, the alteration of certain inflammatory mRNAs helped us understand the pathology of hypertensive stroke.The first part The risk factors for acute hypertensive stroke in the cross-sectional studyObjective:To identify the risk factors for hypertensive intracerebral hemorrhage and hypertensive ischemic stroke compared with hypertensive healthy volunteers, and explore the potential risk factors contribute to hypertensive stroke.Methods:We recruited patients with first-ever IS or ICH attending Department of Neurology, Zhujiang Hospital of Southern Medical University, Guangzhou, China between January 2013 and December 2014. Control subjects without previous cardiovascular and cerebrovascular diseases were selected from Department Cardiology, Zhujiang Hospital of Southern Medical University, Guangzhou, China over the same period. The inclusion criteria were as follows:(1) stoke patients (IS or ICH) were confirmed by magnetic resonance imaging (MRI) or computed tomography (CT) associated with hypertension; (2)the time of symptom onset was within 7 days prior to inclusion; (3) who had no stroke history previously; (4) IS patients didn’t show any evidence of hemorrhagic transformation. The exclusion criteria were:(1) mixed stroke; (2) vascular malformation, aneurysm, atrial fibrillation, vasculitis, rheumatic heart disease, valvular heart disease, coronary heart disease, mental illnesses, autoimmune diseases, malignancies; (3) infections; (4) serious heart, liver or kidney function impairment, blood disorders. Information were retrieved from medical records or via questionnaires or by biomarker assay. It includes demographic variables (e.g. age and sex), cardiovascular risk factors, past medical history, laboratory tests (e.g. blood lipids), the relevant imaging data (e.g. cranial MR and CT, carotid ultrasound and ultrasonic cardiogram) and so on. Hypertension was defined as a diastolic blood pressure≥90mmHg and/or a systolic blood pressure≥140 mmHg, or currently using antihypertensive drugs.Results1. There was fewer people who had efficacious anti-hypertensive therapy in IS and ICH group compared with controls (P,0.002, for IS patients; P,0.015, for ICH patients).2. The presence of left ventricular hypertrophy (OR,5.94; CI,1.30-26.96; P,0.02) and unstable plaque remained (OR,3.33; CI,1.30-8.52; P,0.017) statistically significant for IS after adjusted for confounding factors.Conclusion:1. Antihypertensive therapies and good control of blood pressure had resulted in a reduced incidence of IS and ICH.2. Atherosclerosis and carotid plaque vulnerability were significantly associated with the incidence of hypertensive stroke.3. Left ventricular hypertrophy confers an increased risk of cerebral ischemia.The second part Circulating inflammatory mRNA associated with occurrence and development of acute stroke in patients with hypertensionObjective:To investigate whether atherosclerosis-related mRNAs are correlated with acute stroke in patients with hypertension. The differentially expressed mRNAs in patients with IS or ICH may play a critical role in pathogenesis of hypertensive stroke.Methods1. We recruited patients with first-ever IS or ICH attending Department of Neurology, Zhujiang Hospital of Southern Medical University, Guangzhou, China between January 2015 and December 2015. Control subjects without previous cardiovascular and cerebrovascular diseases were selected from Department Cardiology, Zhujiang Hospital of Southern Medical University, Guangzhou, China over the same period. The detailed inclusion criteria, exclusion criteria, and patients’ basic characteristic referred to the first part.2. Blood samples were collected after an overnight fasting on the second day of hospitalization with EDTA-containing tubes. Total RNA extraction from whole blood was performed using Trizol reagent following blood drawing in two hours. Then the extractive RNA was reverse-transcribed into cDNA. Finally, the synthetic cDN A template was amplified with RT-PCR.3. Compare the expression of MMP9 mRNA, KLF4 mRNA, COL1A2 mRNA and sCD40L mRNA between hypertensive stroke patients and hypertensive healthy subjects. We further investigate whether the differentially expressed mRNAs were related to the instability of carotid plaque.Results:1、The normalized MMP9 mRNA expression was 0.82-fold higher in the ICH group and 0.9-fold higher in the IS group than that in control group (P,0.019, for IS patients; P,0.048, for ICH patients). KLF4 mRNA showed a higher level in patients with cerebral ischemia than that in healthy controls (fold change:1.64, P=0.024).2、The difference of the concentration of KLF4 mRNA and MMP9 mRNA between the plaque group and non-plaque group was not statistically significant, even between stable plaque group and unstable group.Conclusions:1、MMP9 mRNA was significantly correlated with ICH and IS in patients with hypertension. And we first discovered that KLF4 elevated in hypertensive patients with IS.2、Higher levels of KLF4 mRNA and MM9 mRNA maybe not related to carotid plaque vulnerability.