الفهرس 
Introduction and Aim of the WorkOnly 14 pages are availabe for public view
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Abstract
Stroke is the second highest cause of death globally and a leading cause of disability, with an increasing incidence in developing countries. Several phenotypic risk factors are associated with increased possibility of having a stroke. Some are characteristic for each individual, like ethnicity, age, gender, or genetics (non-modifiable risks), and others are attributed to environmental or behavioral factors such as cigarette smoking, hypertension and diabetes mellitus (modifiable risks). Identification of the modifiable stroke risk factors in a certain population might help reduce the burden of stroke in that population.
IS is the most common type of strokes; it results in neurological death and long-term disability which imposes massive health and economic burdens. IS is a polyetiologic disease with distinct differences between subtypes regarding outcome and risk factors. LVD and SVD are two IS subtypes that have long been studied. Though the pathological mechanisms of LVD and SVD are distinct from each other, still both processes could be overlapping and both share similar risk factors, hence, the study of IS as a whole may not be adequate to assess the effects of risk factors under the influence of subtype distribution. Therefore, determining differences in the risk factor profiles among IS subtypes can inform future research for more targeted solutions.
Many studies have tried to elucidate the mechanisms behind SVD; one of the proposed mechanisms is the breakdown of BBB. The integrity of BBB is kept by a complex structure of TJ proteins, of which, CLDN5 and ZO-1 are the key structural and functional components. When BBB is damaged, the TJ proteins CLDN5 and ZO-1 are released from the barrier and become detectable in blood. Circulating TJ proteins have been assessed as biomarkers for BBB disruption in neurologic disorders such as IS.
BBB integrity is also determined by PCs; which secret signals necessary for contractile properties that are essential for regulation of CBF. Contraction and relaxation of PCs contributes to control of CBF. The lack of oxygen after ischemia induces relaxation of PCs which helps dilate blocked vessels and restore CBF. Multiple mediators including PDGF-B, along with others, have been identified as potential regulators of PC contractility after stroke.
Although SVD is one of the major subtypes of stroke, GWAS, have not discovered specific risk loci for SVD. However, in 2016, FOXF2 gene was identified as a novel risk locus for SVD. FOXF2 is expressed in brain PCs, reduction in FOXF2 function has been associated with BBB leakage and deficient production of PDGFRβ.
Another important mechanism implicated in PC development and maintenance is the NOTCH pathway; there is a strong evidence that NOTCH3 promotes expansion of brain PC number by promoting cell division, raising the possibility that NOTCH3 signaling helps determine PC coverage of brain blood vessels.
The aim of the present study was to investigate the prevalence and impact of some stroke risk factors on the levels of ZO-1, CLDN5, and PDGF-B in two IS subtypes; namely: LVD and SVD. This study also aimed to investigate whether FOXF2 and NOTCH3 genes can serve as candidate loci for cerebral SVD susceptibility in Egyptian patients.
This study was approved by the ethical committee of neurology and psychiatry department, faculty of medicine, Ain Shams University. Patients gave their permission for reviewing their medical records and for withdrawal of blood sample. Consent by first degree relative or guardian was obtained for patients who were severely ill or unconscious.
The present study was performed on 186 IS patients. All patients were selected from neurology and psychiatry department in Ain Shams University hospitals. The patients were classified into two groups: group I (n= 114) which included 114 patients with SVD, and group II (n= 72) which included 72 patients with LVD. The diagnosis in both groups was based on clinical, biochemical, MRI and carotid duplex investigations.
A blood sample was collected from each patient and then each blood sample was divided into two portions; one portion was collected on EDTA coated tube for DNA extraction and genotyping studies of FOXF2 rs41300825 (G/C), NOTCH3 rs1043994 (A/G), and the other was collected on serum separating tube for the assay of the different biochemical parameters. The demographic characteristics of each patients’ group were determined and the prevalence of each stroke risk factor was investigated.
Age
¬The mean age of SVD patients was 69.47 ± 0.798 years which was significantly higher than that of the LVD patients; 60.19 ± 1.87 years. Of the SVD patients, 57.89 % were ≥ 70 years old, compared to 25% of the LVD patients, with a significant difference between the two groups (p= 0.031). However, in order to investigate the effect of age as an IS risk factor, each patient group was further subdivided, according to age, into two subgroups; those < 70, and those ≥ 70 years old, and then the prevalence of each stroke risk factor was investigated in each age subgroup. In the LVD group, the mean age of the patients < 70 was 55.96 ± 1.87, while that of the patients ≥ 70 was 72.89 ± 0.807 years old. Hypertension and diabetes mellitus were more prevalent in the ≥ 70 patients. The frequency of hypertension in the patients ≥ 70 was 88.9%, which was significantly higher (p= 0.009) than that in the patients < 70 (37%), likewise, diabetes mellitus was more frequent in the patients ≥ 70, with a frequency of 55.55%, compared to 37.03% in the patients < 70, with a high significant difference between the two groups (p= 0.002). In contrast to hypertension and diabetes mellitus, smoking was more prevalent in the patients < 70; as 55.5% of them were smokers, compared to only 33.3% of the ≥ 70 patients, with a high significant difference between the two groups (p < 0.001), which might explain why 75% of the studied LVD patients were < 70. Meanwhile, 22.2% of the LVD < 70 patients had IHD, while none of the older patients had IHD, with a high significant difference between the two subgroups (p < 0.001).
On the other hand, in the SVD group, the mean age of patients < 70 was 63.5 ± 0.737 which was significantly lower than that of the patients ≥ 70; which was 73.82 ± 0.487 years old. In contrast to LVD patients, the frequency of hypertension in the patients < 70 years (75%) was significantly higher (p < 0.001) than that in the patients ≥ 70 (63.6%). Likewise, diabetes mellitus was more frequent in the patients < 70, with a frequency of 87.5%, compared to only 45.5% in the ≥ 70 patients, with a high significant difference between the two groups (p < 0.001). Concerning smoking, only 12.5% of the patients < 70 were smokers, compared to 18.2 % of the patients ≥ 70, with no significant difference between the two groups. On the other hand, while 25% of the < 70 SVD patients had IHD, 27.3% of the ≥ 70 patients had IHD, with no significant difference between the two groups.
IHD, hypertension, Diabetes mellitus, and smoking
Only 16.7% of the LVD patients and 26.3% of the SVD patients had IHD, with no significant difference between the two groups. In contrast, hypertension and diabetes were prevalent in both groups, being significantly higher in the SVD group. The frequency of hypertension in the SVD group was 68.4%, which was significantly higher (p= 0.04) than that in the LVD patients, which was 50%. Likewise, the frequency of diabetes in the SVD group was 63.2%, which was significantly higher (p= 0.043) than that in the LVD group, which was 41.7%. Significantly (p= 0.001) more smokers were found in the LVD group compared to the SVD group, where the frequency of non-smokers, smokers and heavy smokers in the LVD group was 50%, 16.7% and 33.3%, compared to 84.2%, 10.5%, and 5.3% in the SVD, respectively.
In order to study the impact of each stroke risk factor on the levels of ZO-1, CLDN5 and PDGF-B in each stroke subtype, serum levels of ZO-1, CLDN5 and PDGF-B were compared between LVD and SVD patients, but, unlikely, the comparison did not reveal any significant differences except for PDGF-B, which showed significantly higher (p= 0.04) levels in the LVD group (157.38 ± 18.78), compared to that in the SVD group (114.4 ± 11.3). However, in order to further investigate the changes in the levels of the measured TJ proteins along with PDGF-B in patients with IS, comparisons were made regarding each of the studied IS risk factors.
*Impact of age on serum levels of ZO-1, CLDN5 and PDGF-B
Comparing serum levels of ZO-1, CLDN5 and PDGF-B between stroke patients < 70 and ≥ 70 years old revealed significant differences only in the levels of ZO-1, which was significantly higher (p= 0.03) in patients < 70 years (11.708 ± 1.21) compared to those ≥ 70 years patients (8.18 ± 0.66). The same comparison between LVD patients < 70 and ≥ 70 years old did not reveal any significant differences. Meanwhile, SVD patients < 70 patients had significantly (p= 0.008) higher levels of PDGF-B than those ≥ 70, with levels of (149.09 ± 22.81) and (89.16 ± 8.17), respectively.
*Impact of diabetes on serum levels of ZO-1, CLDN5 and PDGF-B
Comparing serum levels of ZO-1, CLDN5 and PDGF-B between diabetic and non-diabetic stroke patients did not reveal any significant differences. The same comparison between diabetic and non-diabetic LVD patients revealed significantly (p= 0.018) higher levels of ZO-1in the non-diabetic LVD patients (13.74 ± 1.59) compared to the diabetics (8.87 ± 0.92).
On the other hand, SVD patients surprisingly revealed significantly (p= 0.017) higher levels of ZO-1in the diabetic patients (11.59 ± 1.69) compared to the non-diabetics (6.08 ± 0.27). This might be attributed to age, as diabetes mellitus was more frequent in the patients < 70, with a frequency of 87.5%, compared to 45.5% in the ≥ 70 patients, with a high significant difference between the two groups (p < 0.001).
*Impact of IHD on serum levels of ZO-1, CLDN5 and PDGF-B
Comparing serum levels of ZO-1, CLDN5 and PDGF-B between patients with and without IHD revealed significantly higher levels of: * ZO-1, CLDN5 and PDGF-B in non-IHD stroke patients compared to those with IHD; *ZO-1 and PDGF-B in non-IHD-LVD patients compared to IHD-LVD patients, in addition to * ZO-1 and CLDN5 in non-IHD-SVD patients compared to those with IHD.
*Impact of smoking on serum levels of ZO-1, CLDN5 and PDGF-B
Comparing serum levels of ZO-1, CLDN5 and PDGF-B between smoker and non-smoker stroke patients revealed significantly (p= 0.019) higher levels of ZO-1 in the non-smokers (14.98 ± 4.06), compared to smokers (9.28 ± 0.71). CLDN5 levels were also significantly (p= 0.02) higher in the non-smoker stroke patients (7.78 ± 0.49), compared to the smoker patients (4.89 ± 0.66). The same comparison between smoker and non-smoker LVD and SVD patients did not reveal any significant difference.
*Impact of hypertension on serum levels of ZO-1, CLDN5 and PDGF-B
Comparing serum levels of ZO-1, CLDN5 and PDGF-B between normotensive and hypertensive stroke patients revealed significantly higher levels of ZO-1 and CLDN5 in the normotensive patients compared to hypertensive patients. The same comparison between normotensive and hypertensive LVD patients did not reveal any significant differences, while in SVD patients, it revealed significantly (p= 0.037) higher levels of ZO-1 in the normotensive patients (13.007 ± 3.09) compared to hypertensive patients (7.97 ± 0.73).
*Role of single nucleotide polymorphisms in susceptibility of stroke
In the present study, FOXF2 rs41300825 could not be correlated with neither the SVD nor the LVD group, as the results of FOXF2 rs41300825 genotyping showed an amplified PCR product of 334 bp, representing only one genotype; (GG), which was further confirmed by DNA sequencing.
The same was true for NOTCH3 rs1043994, as the results of NOTCH3 rs1043994 genotyping demonstrated an amplified PCR product of 656 bp, representing only one genotype; (GG), which was further confirmed by DNA sequencing.