Sublingual Microcirculation ——Characteristics and Determinants in Chinese and Flemish Populations Yan Li MD PhD Shanghai Institute of Hypertension

Hypertension and Microcirculation Impaired vasodilation … Functional rarefaction Hypertension Structural rarefaction Apoptosis in endothelial cells

Vascular Glycocalyx Glycocalyx consists of proteoglycans and glycosaminoglycans. This the picture of the glycocalyx under the electron microscope. The glycocalyx forms a gel-like layer protecting the endothelial lining of the arteries. The glycocalyx plays a role in the balance of vasoconstriction and vasodilatation, pro- and anti-coagulant activity, pro- and anti-inflammatory stimuli, and pro- and anti-oxidative factors The glycol-calyx plays an important role in maintaining the balance between vasoconstriction and vasodilatation, pro- and anti-coagulant activity, pro- and anti- inflammatory stimuli, and pro- and anti-oxidative factors. Most studies published to date had a small sample size or a case–control design, no previous population study focused on the sublingual microcirculation to assess capillary density and glycol-calyx width The glycocalyx is a gel-like layer about 0.5 μm thick covering the endothelium. It serves as a physical barrier and plays an important role in vascular hemeostasis.

Sublingual Capillary Microscopy and GlycoCheck 1 site=10micros, need not say first mucosa[mju:'kəʊsə] Handheld Video Capillary Microscope (KK Research Technology, UK) interfaced with a computer running the GlycoCheck software (GlycoCheck BV, Maastricht, The Netherlands).

GlycoCheck Measurement 1 ― Capillary Density Total density (RBC filling >0%) no segments / mm2 Perfused density (RBC filling ≥50%) Perfused-to-total density ratio Quality control is by the software The vertical lines are the reference lines used by the software at intervals of 10-um only choose the segment perfused by the blood flow>=50% 0.4 mm

GlycoCheck Measurements 2 ― Perfused Boundary Region (PBR) RBC column width at the 3000 capillary segments 10 um apart was determined and the median and the 5th-95th percentile interval of RBC width was used for the calculation of PBR.

Healthy and Impaired Glycocalyx as Measured by PBR

Sublingual Glycocheck Previous Studies with Sublingual Glycocheck The sublingual glycocalyx estimated by the PBR is decreased in patients with DM, CKD, CVD, and critically ill patients. Limitations: case-control studies; small sample size; no population data Glycocalyx consists of proteoglycans and glycosaminoglycans. This the picture of the glycocalyx under the electron microscope. The glycocalyx forms a gel-like layer protecting the endothelial lining of the arteries. The glycocalyx plays a role in the balance of vasoconstriction and vasodilatation, pro- and anti-coagulant activity, pro- and anti-inflammatory stimuli, and pro- and anti-oxidative factors

Study Objective We investigated the characteristics and associations of the sublingual capillary density and glycocalyx with common CV risk factors in general Chinese and Flemish populations. Gu YM, et al. Hypertension. 2015;65:993-1001.

Study Populations ― Flemish From April 2013 to June 2014, 487 participants were invited, 320 signed informed consent(65.7%). The number of participants analyzed totaled 254. 600km far away from shanghai Altitude: average 600m Eksel, Belgium Coordinating Center in Leuven, Beglium

Study Populations ― Han and She Chinese In May 2013, 931 Chinese were invited, 663 (71.2%) gave informed consent. The number of participants analyzed totaled 472, including 252 Han and 220 She people. 600km far away from shanghai Altitude: average 600m JingNing County Villages in JingNing She Minority

Measurements in the 3 Ethnic Groups Sublingual GlycoCheck Standardized questionnaires Anthropometric measurements Blood biochemical measurements 600km far away from shanghai Altitude: average 600m

Characteristics of Participants (1) Han (n=252) She (n=220) Flemish (n=254) Age, y 51.8 51.7 49.8 BMI, kg/m2 23.2‡ 23.5† 26.5 SBP/DBP, mmHg 124/78*‡ 129/81 129/83 PP, mm Hg 46.8 47.6 46.5 Pulse rate, beats/min 72.1‡ 73.2† 63.3 Hematocrit, % 43.3‡ 42.7† 41.1 Total cholesterol, mmol/L 5.40‡ 5.57† 4.82 Blood glucose, mmol/L 4.99‡ 4.79† 4.57 Significance of the difference: * Han vs. She, † She vs. Flemish, ‡ Han vs. Flemish.

Characteristics of Participants (2) Han (n=252) She (n=220) Flemish (n=254) Women 55.6 62.7† 46.9 Smokers 21.8 17.3 14.2 Drinking alcohol 34.1‡ 31.4† 76.4 HT (Anti-HT) 29 (42) 36 (48) 38 (44) Previous CVD 3.2 1.8 5.5 Diabetes 6.4‡ 0.4 Significance of the difference: * Han vs. She, † She vs. Flemish, ‡ Han vs. Flemish.

GlycoCheck Measurements Han (n=252) She (n=220) Flemish (n=254) PBR, nm 2019‡ 2000† 1876 Total Capillary density, NO/mm2 580‡ 573† 546 Perfused Capillary density, NO/mm2 342‡ 334 320 Perfused-to-total density ratio 0.59 Significance of the difference : * Han vs. She, † She vs. Flemish, ‡ Han vs. Flemish.

PBR, Hemotocrit and Capillary Density Han 2700 2500 2300 2100 1900 1700 1500 30 35 40 45 55 50 600 500 400 300 200 100 Perfused density (n°/mm2) Haematocrit (%) PBR (nm) p<0.0001 She Perfused density (n°/mm2) 2700 2500 2300 2100 1900 1700 1500 30 35 40 45 55 50 600 500 400 300 200 100 Haematocrit (%) p<0.0001 Flemish 2500 2300 2100 1900 1700 1500 1200 30 35 40 45 55 50 600 500 400 300 200 100 Perfused density (n°/mm2) Haematocrit (%) P<0.0001 Association of the perfused boundary region with hematocrit and perfused capillary density by ethnicity. The scale of hematocrit increases from back to front, whereas the opposite is the case for perfused capillary density.

Distribution of Standardized PBR

Standardized PBR P<0.0001 Standardized PBR (nm) P<0.0001 2024 1990 1875

Correlates of Standardized PBR Model 1 Model 2 Model 3 Han (vs. average) 50.4* 49.3* 53.6* She (vs. average) 24.3 24.6 20.8 Flemish (vs. average) –74.7* –73.9* –74.5* BMI (+3.9 kg/m2) –18.4 –17.4 –25.3* MAP (+13.1 mmHg) –23.3* DBP (+11.2 mmHg) –25.4* The covariables considered were sex, age, BMI, smoking, cholesterol, glucose, g-GT, AH-drug, CVD, and MAP and PP in Model 1, or SBP and DBP in Model 2. or Framingham risk score in Model 3. Significance of effect size: * P≤0.05.

Correlates of Standardized PBR by Ethnicities BMI MAP None of the between-ethnicity comparisons reached significance.

Correlates of Perfused Capillary Density 80 Regression line (95% CI) in individual participants Mean (95%CI) per quintile Perfused density (n°/mm2) 30 40 50 60 70 p=0.0024 year 375 350 325 300 Age p=0.009 –8 –4 4 8 point 12 Framingham score p=0.45 p=0.14 15 20 25 35 kg/m2 3 5 6 7 mmol/L BMI Cholesterol Perfused capillary density was standardised for ethnicity.

Correlates of Capillary Density Perfused Density Total density Model 1 Model 2 Han (vs. average) 10.0* 9.29* 15.2* 13.5* She (vs. average) 1.58 1.49 6.65 6.59 Flemish (vs. average) –11.6* –10.8* –21.8* –20.1* Age (+13.7y) 9.31† … Framingham score (+6.53 pts) 7.93† Correlates of the sublingual capillary density were identified by a stepwise regression procedure with P-values for covariables to enter and stay in the models set at 0.15. Ethnicity was forced in the models and was coded using the deviation from mean coding, which expresses the ethnic differences relative to the average in the whole study population. The covariables considered for entry in Models 1 were female sex (0, 1), age (continuous), body mass index (continuous), smoking (0, 1), total cholesterol (continuous), plasma glucose (continuous), g-glutamyltransferase (continuous), antihypertensive drug treatment (0, 1), and history of cardiovascular disease (0, 1). Mean arterial pressure and pulse pressure, or systolic and diastolic blood pressures were also offered to Model 1, but did not enter. Covariables in Model 2 were body mass index, g-glutamyltransferase, Framingham risk score, antihypertensive drug treatment, and history of cardiovascular disease. For continuous variables, the association sizes are expressed for a 1‑SD increase. Significance of the associations: * P≤0.05; †P≤0.01; ‡P≤0.001. The covariables considered were sex, age, BMI, smoking, cholesterol, glucose, g-GT, AH-drug, CVD. Model1 additionally included MAP and PP, whereas Model 2 had Framingham risk score. Significance of effect size: * P≤0.05. 22

Correlates of Perfused-to-Total Capillary Density Ratio Model 1 Model 2 Han (vs. average) 0.0060 0.0050 She (vs. average) –0.0051 –0.0027 Flemish (vs. average) –0.0009 –0.0023 Age (+14.4 y) 0.0092* BMI (+3.90 kg/m2) 0.0085* 0.0066 Cholesterol (+1.02mm/L) 0.0102* Smoking (0,1) –0.0202* Framingham risk score (+6.53pts) 0.0145† We applied the deviation from mean coding, which expresses the ethnic differences in the ratio relative to the average in the whole study population. 23

Correlates of Perfused-to-Total Capillary Density Ratio by Ethnicities Age BMI TC FRS None of the between-ethnicity comparisons reached significance.

Summary Higher age, body mass index, total cholesterol and Framingham risk score were associated with functional capillary recruitment. In capillaries that remain perfused, glycocalyx width increased with higher BMI, MAP and DBP. There were no ethnic differences in these associations.

Conclusion A higher cardiovascular risk profile is associated with recruitment of capillaries with preserved glycocalyx that protects the endothelium. Further research on microcirculation with the use of this novel technique is warranted.

Acknowledgements Leuven Shanghai Jan A. Staessen Yumei Gu Yanping Liu Lutgarde Thijs Tatiana Kuznetsova Peter Verhamme Thibault Petit Zhengyu Zhang Fangfei Wei Shanghai Jiguang Wang Yan Li Changsheng Sheng Qifang Huang Shuai Wang Yuanyuan Kang Feika Li Jie Song Yibang Cheng Qianhui Guo

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