Peripheral microvascular dysfunction is also present in patients with ischemia and no obstructive coronary artery disease (INOCA)

Abstract

BACKGROUND:

In patients with ischemia and no obstructive coronary artery disease (INOCA), coronary microvascular dysfunction is associated with higher rate of major adverse cardiovascular events.

OBJECTIVE:

To demonstrate if microvascular dysfunction present in coronary microcirculation of patients with INOCA may be detected noninvasively in their peripheral circulation.

METHODS:

25 patients with INOCA and 25 apparently healthy individuals (controls) were subjected to nailfold videocapillaroscopy (NVC) and venous occlusion plethysmography (VOP) to evaluate peripheral microvascular function and blood collection for biomarkers analysis, including soluble vascular cell adhesion molecule-1 (sVCAM-1), endothelin-1 (ET-1) and C-reactive protein (CRP).

RESULTS:

Red blood cell velocity (RBCV) before and after ischemia (RBCV_max) were significantly lower in patients with INOCA (p = 0.0001). Time to reach maximal red blood cell velocity (TRBCV_max) was significantly longer in INOCA group (p = 0.0004). Concerning VOP, maximal blood flow (p = 0.004) and its relative increment were significantly lower in patients with INOCA (p = 0.0004). RBCV_max showed significant correlations with sVCAM-1 (r = –0.38, p < 0.05), ET-1 (r = –0.73, p < 0.05) and CRP (r = –0.33, p < 0.05). Relative increment of maximal post-ischemic blood flow was significantly correlated with sVCAM-1 (r = –0.42, p < 0.05) and ET-1 (r = –0.48, p < 0.05).

CONCLUSIONS:

The impairment of microvascular function present in coronary microcirculation of patients with INOCA can be also detected in peripheral microcirculation.

Keywords

Ischemia and no obstructive coronary artery disease microvascular dysfunction nailfold videocapillaroscopy venous occlusion plethysmography

1 Introduction

The historical term Cardiac Syndrome X (CSX), firstly introduced by Kemp [1] corresponds to a heterogenous group of patients that presents typical angina and angiographically normal epicardial arteries [2, 3]. Due to stigmatization by its associations with female gender, obesity, and psychology, recently the term CSX has been replaced by INOCA (ischemia and no obstructive coronary artery disease) [4]. This new term refers to a syndrome that encompasses all patients with symptoms and signs of myocardial ischemia in the absence of coronary artery disease (CAD) [5]. In patients with INOCA, myocardial ischemia can be detected by noninvasive tests, such as myocardial perfusion single-photon emission computed tomography (SPECT) imaging.

The etiology of myocardial ischemia (i.e. the mismatch between blood supply and myocardial oxygen demand) in patients with INOCA involves coronary microvascular dysfunction (CMD) and/or epicardial coronary artery spasm [6]. CMD may be structural and functional or both [7]. Structural alterations in CMD encompasses microvascular remodeling, including reduction in capillary lumen and in capillary number (i.e. rarefaction), which increases coronary microvascular resistance. Functional abnormalities involve enhanced vasoconstriction and impaired vasodilation as consequence of endothelium-dependent and independent mechanisms or a combination of both [8].

The reduction of both endothelium dependent or/and independent function [9] and both are associated with worse outcomes [10]. Coronary endothelium independent dysfunction is characterized by a decreased coronary flow reserve (CFR) [11]. CFR is defined as the ratio of hyperemic blood flow in response to various vasoactive stimuli divided by resting blood flow [7] and is the clinical reference standard for quantitative evaluation of microvascular vasodilatory reserve [12].

Since microvascular dysfunction has been considered a systemic process [13, 14], we have hypothesized that microvascular dysfunction, reflected by reduced hyperemic response, observed in coronary microcirculation of patients with INOCA may be also detected in peripheral microcirculation of these patients.

2 Methods

This is a cross-sectional study, conducted from March 2014 to August 2015, approved by the Ethics Committee of the Hospital Universitário Pedro Ernesto, Universidade do Estado do Rio de Janeiro (CAAE 08993412.8.1001.5259) and performed according to principles outlined in the Declaration of Helsinki (ClinicalTrials.gov Identifier NCT03243968). Local ethical approval was obtained prior to study start and all participants provided their written informed consent.

2.1 Subjects

The microvascular function of 25 patients diagnosed with INOCA (INOCA group), which present normal coronary angiography and myocardial ischemia detected by Technecium (Tc99m) gated SPECT, were compared to 25 apparently healthy individuals (control group, CG). The CG participants did not present any signs, symptoms and risk factors for cardiovascular disease that could justify the indication for angiography and SPECT and, therefore, they were not subjected to these exams. The INOCA group was recruited from Departamento de Cardiologia do Hospital Universitário Pedro Ernesto, Universidade do Estado do Rio de Janeiro and both groups underwent cardiovascular risk assessment using Framingham Coronary Heart Disease Risk Score [15].

Instead of performing sample size calculation, we established a period for participants inclusion (March 1st, 2014 to August 30th, 2015). All patients with diagnostic of INOCA and control group participants were included in the study during this period.

2.2 Inclusion criteria

Men or women with or without INOCA, over 18 years old, body mass index (BMI) < 35 kg/m², able to follow the given instructions and to evaluate microvascular function.

2.3 Exclusion criteria

We have excluded subjects with diagnosis of diabetes mellitus and heart failure, defined according to American Diabetes Association (ADA) [16] and Brazilian Guideline of Acute and Chronic Heart Failure [17] criteria, respectively. In addition, individuals with chronic kidney disease [18] and recent history of myocardial infarction (period inferior to six months), stroke and trauma, cancer, autoimmune diseases, infectious diseases, obesity (BMI ≥35 kg/m²), hypertension (in the case of control group), uncontrolled and resistant hypertension (in the case of INOCA group) and users of anti-inflammatory drugs were excluded from the study.

2.4 Coronary angiography and Tc99m gated SPECT imaging

Coronary angiography was performed to identify critical obstructive epicardial coronary arteries. In the absence of CAD (i.e. in the case of angiographically smooth unobstructed arteries) the patients underwent Tc99m gated SPECT imaging to evaluate myocardial perfusion at rest and after exertional stress on treadmill. Tc99m Sestamibi (Cardiolite, Bristol-Myers Squibb Medical Imaging, Inc., MA, USA) was administered intravenously before and after cardiologic stress to allow the comparisons of images between these two timepoints in terms of Tc99m Sestamibi myocardial uptake. The microvascular dysfunction in patients with INOCA and its consequent myocardial hypoperfusion is evidenced by decreased uptake of this radioisotopic tracer after exercise stress test in comparison to rest condition.

2.5 Evaluation of peripheral microvascular function

All participants were asked to arrive at the laboratory after 8 h overnight fast and to abstain from caffeine and alcohol for 24 h. They were accommodated in an acclimatized room (24±1°C) during 30 min before evaluations.

All subjects had their anthropometric variables recorded and blood pressure evaluated before examinations. For cardiovascular risk evaluation, participants filled a cardiovascular risk assessment questionnaire. For microvascular evaluations, venous occlusion plethysmography (VOP) and nailfold videocapillaroscopy (NVC) techniques were used.

NVC is a noninvasive technique used to assess nutritional blood supply in human skin capillaries. Using NVC, capillaries can be seen parallel to the skin [19], allowing capillary morphological and functional evaluations [20, 21].

VOP is used to investigate peripheral microvascular function through assessment of blood flow increase in response to ischemia, i.e. reactive hyperemia. VOP also assesses vascular smooth muscle cell (VSMC) integrity, through evaluation of vasodilation elicited by an exogenous donor of nitric oxide (NO).

2.6 Functional assessment of peripheral microcirculation using Venous Occlusion Plethysmography (VOP)

Firstly, a pressure cuff was placed above the antecubital fossa of the left arm of the participant and positioned on a support that ensured that the arm was set at heart level. Blood pressure was measured in the contralateral arm and heart rate was continuously monitored by electrodes placed on the chest connected to the plethysmograph (Hokanson®AI6 plethysmograph, AD Instruments, Castle Hill, NSW, Australia). Forearm volume alterations were detected by a mercury and silicone tape-shaped sensor that surrounded the forearm at its greatest diameter. The observed changes in forearm volume were recorded as variations of vascular resistance and mathematically converted into arterial blood flow in mL/min/100 mL of tissue by the plethysmograph analysis software. For maintenance of linear correlation between forearm arterial blood flow and its volume increase, the forearm circulation was isolated from the hand circulation through a cuff placed around the wrist inflated above systolic blood pressure.

VOP was performed in four sequential steps, with minimum intervals of three minutes. Immediately before each step, the blood pressure of the participants was measured. The four steps were: 1) Baseline arterial blood flow 1 –in this step, four measurements of arterial blood flow without any external stimulus were performed and the mean of three measurements with the lowest standard deviation was considered; 2) reactive hyperemia –in this step, the arm cuff was inflated to 200 mmHg for 5 min to induce forearm ischemia [22] and then deflated. Within 10 s, the cuff was inflated again to 50 mmHg. This pressure, higher than venous pressure and lower than diastolic blood pressure, causes an impairment of venous return and allows the increase of forearm blood volume as consequence of ischemia release. This vasodilation in response to ischemia release is called reactive hyperemia. Forearm arterial blood flow was registered during the reactive hyperemia and was considered the mean of four observed arterial blood flow measurements and the maximal value of arterial blood flow; 3) baseline arterial blood flow 2 –arterial blood flow measurements were performed after 15 min of rest to completely wash out any vasoactive factor, released during ischemia, that could interfere in the analysis afterwards. In this step, four measurements were performed and the mean of three measurements with the lowest standard deviation was considered and; 4) post-nitroglycerin –in this step, forearm arterial blood flow 5 min after 400μg of sublingual nitroglycerin (Nitrolingual Burns Adler Pharmaceuticals, Inc., Charlotte, NC, USA) application were assessed. Nitroglycerin is an exogeneous nitric oxide donor used to verify vascular wall integrity by means of endothelial independent vasodilatation. The mean of four observed arterial blood flow measurements and the maximal value of arterial blood flow were considered.

During hyperemia and post-nitroglycerin steps, increments of arterial blood flow during reactive hyperemia response and post nitroglycerin application were expressed in absolute values (difference between maximal and mean arterial blood flow after ischemia and after nitroglycerin application and mean baseline arterial blood flows 1 and 2, respectively) and in relative values (% of increase from mean baseline arterial blood flows 1 and 2 to mean and maximal arterial blood flow after ischemia and after nitroglycerin application). These values were compared between controls and patients with INOCA.

2.7 Structural and functional assessment of microcirculation using Nailfold Videocapillaroscopy (NVC)

In order to assess microvascular morphology and function, participants placed the fourth finger of the left hand on a platform fixed to an intravital microscope (Leica MZFLIII, Wetzlar, Germany) equipped with epi-illumination (Leica GLS 100, Wetzlar, Germany) and coupled to a videocamera (TK-S250, JVC, Japan). The nailfold microcirculation images were registered by a DVD recorder and thereafter analyzed using the Cap-Image v7.2 software (Zeintl, Heidelberg, Germany).

NVC was performed according to previously standardized methodology in our laboratory [19 –21]. The technique used allows the following functional variables evaluations: Functional capillary density (FCD) i.e. number of perfused capillaries per mm², red blood cell velocity at rest (RBCV) and its peak during the post-occlusive reactive hyperemia (PORH) response after 1 min ischemia (RBCV_max) and time to reach it (TRBCV_max) during PORH. The morphological variables evaluated were: Afferent (AFD), apical (APD) and efferent (EFD) capillary loop diameters. For functional and morphological evaluations, X250 and X680 magnifications were used, respectively.

2.8 Endothelial injury/inflammatory biomarkers analysis

Venous blood was harvested into EDTA tubes to determine soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), adiponectin, endothelin-1 (ET-1), and oxidized low density lipoprotein (oxLDL) plasma concentrations in participants. Plasma levels of sICAM-1 and sVCAM-1 were assessed by Human Cardiovascular Disease (CVD2) Panel 2 Magnetic Bead Kit (EMD Millipore Corporation, MN, USA). Adiponectin, ET-1 and oxLDL plasma concentrations were evaluated by Human Adipokine Magnetic Bead 1 (EMD Millipore Corporation, MN, USA), Human Angiogenesis/Growth Factor Magnetic Bead Panel 1 (EMD Millipore Corporation, MN, USA) and Mercodia oxidized LDL ELISA (Mercodia, Uppsala, Sweden), respectively. Serum samples were also used for High Sensitivity C-Reactive Protein (hs-CRP) analysis using a kit for latex turbidimetric method (Biosystems S.A., Barcelona, Spain).

All assays were performed according to protocols provided by kits manufacturers.

2.9 Statistical analysis

For statistical analysis, the SAS® System, version 6.11 software (SAS Institute, Inc.) was used. Normal Gaussian distribution was checked using Shapiro-Wilk test. Categorical variables were expressed as percentage. Between groups comparisons for continuous and categorical variables were performed by Mann Whitney U and Fisher exact test or χ², respectively. All continuous variables were non-parametrical and expressed as median [interquartile range (IQR)]. The relationship between NVC and VOP variables, evaluated between groups, were analyzed by Analysis of Covariance (ANCOVA) and adjusted by age and BMI. In this analysis, was applied a logarithmic transformation (natural logarithm) in NVC and VOP data. Correlations between NVC and VOP variables and biomarkers concentrations of all participants were performed using Spearman test. Values of p < 0.05 were considered statistically significant.

3 Results

3.1 Anthropometric characteristics and cardiovascular risk assessment of control and INOCA groups

The anthropometric characteristics of both groups are shown on Table 1. The INOCA group presented significantly higher age (p = 0.0003), BMI (p = 0.013), waist circumference (p = 0.001), waist-to-hip ratio (p = 0.008) and fat mass (p = 0.0006) in comparison to control group. Moreover, muscle mass in INOCA group was significant lower in relation to control group (p = 0.0006). No significant differences between groups were observed in relation to height (p = 0.065), weight (p = 0.20) and hip circumference (p = 0.13).

Table 1
Anthropometric characteristics of Control and INOCA groups

Variable Control group INOCA group p value

Age (y) 33.0 [27.5–52.5] 55.0 [49.5–62.0] 0.0003

Height (m) 1.68 [1.63–1.74] 1.62 [1.56–1.71] 0.065

Weight (kg) 71.0 [61.4–78.2] 72.6 [66.0–89.7] 0.20

BMI (kg/m²) 24.5 [23.1–26.9] 29.1 [24.2–30.4] 0.013

WC (cm) 85.0 [73.5–91.3] 97.0 [87.3–103.8] 0.001

HC (cm) 100 [96–104] 105 [96–111] 0.13

WHR 0.87 [0.75–0.91] 0.90 [0.85–0.97] 0.008

Muscle mass (%) 74.7 [71.0–80.1] 70.0 [62.0–72.2] 0.0006

Fat mass (%) 25.3 [20.0–29.1] 30.0 [27.8–38.0] 0.0006

Variable	Control group	INOCA group	p value
Age (y)	33.0 [27.5–52.5]	55.0 [49.5–62.0]	0.0003
Height (m)	1.68 [1.63–1.74]	1.62 [1.56–1.71]	0.065
Weight (kg)	71.0 [61.4–78.2]	72.6 [66.0–89.7]	0.20
BMI (kg/m²)	24.5 [23.1–26.9]	29.1 [24.2–30.4]	0.013
WC (cm)	85.0 [73.5–91.3]	97.0 [87.3–103.8]	0.001
HC (cm)	100 [96–104]	105 [96–111]	0.13
WHR	0.87 [0.75–0.91]	0.90 [0.85–0.97]	0.008
Muscle mass (%)	74.7 [71.0–80.1]	70.0 [62.0–72.2]	0.0006
Fat mass (%)	25.3 [20.0–29.1]	30.0 [27.8–38.0]	0.0006

Data are expressed as median [interquartile range]. INOCA Group- Ischemia with no obstructive coronary artery disease group. BMI–body mass index. WC–waist circumference. HC-hip circumference. WHR-waist-to-hip ratio.

INOCA group presented significantly greater number of individuals with definite angina (substernal discomfort that was precipitated by exertion and relieved by rest or nitroglycerin in less than 10 minutes) [23], arterial hypertension (systolic blood pressure≥140 mmHg and diastolic blood pressure≥90 mmHg), dyslipidemia (blood levels of low density lipoprotein (LDL) > 130 mg/dl, triglycerides > 150 mg/dl and high density lipoprotein (HDL) < 40 mg/dl after 12 h fast), mental stress (presence of two or more of the following conditions: 1-anxiety, anguish, nervousness or excessive worry; 2-irritation and impatience; 3-dizziness; 4-concentration and memory problems, 5-loss of control sensation; 6-difficult to sleep), family history of CAD (presence of familiar history of CAD in men below 55 years old and women below 65 years old), sedentarism (practice of physical activity inferior to 150 min per week), smoking habit (individual who has smoked more than 100 cigarettes, or 5 packs of cigarettes, in his entire life or currently smokes any quantity), and a significantly smaller number of physically active (practice of physical activity superior to 150 min per week) subjects compared to control group (Table 2).

Table 2

Cardiovascular risk comparison between control and INOCA groups

	Control group	INOCA group	p value
Female/Male ratio	12/13	15/10	0.57
Caucasian	19	21	0.48
Definite angina¹	0	10	< 0.0001
Physical activity²	17	8	0.011
Arterial hypertension³	0	17	< 0.0001
Dyslipidemia⁴	0	13	< 0.0001
Mental stress⁵	0	17	< 0.0001
Cigarette smoking⁶	0	4	< 0.0001
Family story of coronary artery disease⁷	8	18	< 0.005
Sedentarism⁸	4	9	0.10

All data are expressed as number of patients. Total participants per group = 25. INOCA Group- Ischemia with no obstructive coronary artery disease group. 1-substernal discomfort that was precipitated by exertion and relieved by rest or nitroglycerin in less than 10 minutes. 2- practice of physical activity superior to 150 min per week. 3-systolic blood pressure≥140 mmHg and diastolic blood pressure≥90 mmHg. 4-blood levels of low-density lipoprotein (LDL) > 130 mg/dl, triglycerides > 150 mg/dl and high-density lipoprotein (HDL) < 40 mg/dl after 12 h fast. 5- presence of two or more of the following conditions: a-anxiety, anguish, nervousness, or excessive worry; b- irritation and impatience; c-dizziness; d- concentration and memory problems, e- loss of control sensation; f-difficult to sleep. 6-individual who has smoked more than 100 cigarettes, or 5 packs of cigarettes, in his entire life or currently smokes any quantity. 7- presence of familiar history of Coronary artery disease in men below 55 years old and women below 65 years old. 8- practice of physical activity inferior to 150 min per week.

The cardiovascular risk assessment was performed using Framingham Coronary Heart Disease Risk Score, which estimates the risk of developing CAD within 10 years. Patients with INOCA and controls presented 6% and 2% of being at this risk, respectively. Although, both groups presented low risk for CAD (< 10%), patients with INOCA have three times higher risk than controls.

3.2 Functional assessment of peripheral microcirculation using Venous Occlusion Plethysmography (VOP)

Differences in forearm blood flow between controls and patients with INOCA before and after 5-minutes forearm ischemia are shown on Table 3. Before ischemia, mean baseline flow (p = 0.054) was not significantly different between control and INOCA groups. Regarding mean blood flow (p = 0.42) after ischemia i.e. during the reactive hyperemia response, no significant difference was observed between groups. On the other hand, maximal blood flow (p = 0.004) after ischemia was significantly different between the two groups. Post ischemic relative increment of mean blood flow (p = 0.005) was also significantly different between them. The relative (p = 0.0004) increment of maximal blood flow after ischemia was significantly different between groups.

Table 3
Differences of forearm arterial blood flow between controls and patients with INOCA before and after ischemia and before and after sublingual application of nitroglycerin

Variable Control group INOCA group p value

Blood flow before ischemia

Mean baseline blood flow 1 (mL/min/100 mL of tissue) 1.44 [1.08–2.25] 2.14 [1.40–2.71] 0.054

Blood flow after ischemia

Mean post-ischemia blood flow (mL/min/100 mL of tissue) 6.52[4.70–10.79] 6.03[4.42–8.11] 0.42

Relative increment (%) 346 [267–484] 217 [127–329] 0.005

Maximal post-ischemia blood flow (mL/min/100 mL of tissue) 11.5 [9.52–18.86] 9.27 [7.03–11.65] 0.004

Relative increment (%) 726 [507–1042] 397 [188–571] 0.0004

Blood flow before nitroglycerin

Mean baseline blood flow 2 (mL/min/100 mL of tissue) 1.22 [0.95–1.67] 1.68 [0.89–2.31] 0.18

Blood flow after nitroglycerin

Mean post-nitroglycerin blood flow (mL/min/100 mL of tissue) 1.43 [1.01–1.80] 1.46 [0.98–1.98] 0.64

Relative increment (%) 4.00 [–14.60–39.30] –14.7 [–27.20–27.8] 0.15

Maximal post-nitroglycerin blood flow (mL/min/100mL of tissue) 1.84 [1.35–2.73] 1.91 [1.39–2.70] 0.76

Relative increment (%) 46.90 [8.1–102.9] 15.30 [–1.90–67.3] 0.23

Variable	Control group	INOCA group	p value
Blood flow before ischemia
Mean baseline blood flow 1 (mL/min/100 mL of tissue)	1.44 [1.08–2.25]	2.14 [1.40–2.71]	0.054
Blood flow after ischemia
Mean post-ischemia blood flow (mL/min/100 mL of tissue)	6.52[4.70–10.79]	6.03[4.42–8.11]	0.42
Relative increment (%)	346 [267–484]	217 [127–329]	0.005
Maximal post-ischemia blood flow (mL/min/100 mL of tissue)	11.5 [9.52–18.86]	9.27 [7.03–11.65]	0.004
Relative increment (%)	726 [507–1042]	397 [188–571]	0.0004
Blood flow before nitroglycerin
Mean baseline blood flow 2 (mL/min/100 mL of tissue)	1.22 [0.95–1.67]	1.68 [0.89–2.31]	0.18
Blood flow after nitroglycerin
Mean post-nitroglycerin blood flow (mL/min/100 mL of tissue)	1.43 [1.01–1.80]	1.46 [0.98–1.98]	0.64
Relative increment (%)	4.00 [–14.60–39.30]	–14.7 [–27.20–27.8]	0.15
Maximal post-nitroglycerin blood flow (mL/min/100mL of tissue)	1.84 [1.35–2.73]	1.91 [1.39–2.70]	0.76
Relative increment (%)	46.90 [8.1–102.9]	15.30 [–1.90–67.3]	0.23

Data are expressed as median [interquartile range]. INOCA Group- Ischemia with no obstructive coronary artery disease group.

Differences in forearm blood flow between controls and patients with INOCA before and after sublingual application of nitroglycerin are depicted on Table 3. Before nitroglycerin application, mean baseline flow (p = 0.18) was not significantly different between control and INOCA groups. Regarding mean (p = 0.64) and maximal blood flow (p = 0.76) after nitroglycerin application, no significant difference was observed between groups. Similarly, relative increments of mean (p = 0.15) and maximal (p = 0.23) blood flow after nitroglycerin application were not significantly different between groups.

3.3 Structural and functional assessment of microcirculation using Nailfold Videocapillaroscopy (NVC)

Differences in microvascular morphology and function between control and INOCA groups are shown on Table 4. Patients with INOCA presented significantly greater FCD compared to controls (p = 0.026). Furthermore, RBCV (p = 0.0001) and RBCV_max (p = 0.0001) in patients with INOCA were significantly lower than in controls TRBCV_max in INOCA group was significantly longer than in control group (p = 0.0004). No significant difference between groups could be observed regarding AFD (p = 0.19), APD (p = 0.68) and EFD (p = 0.075).

Table 4
Differences of capillary morphological and functional variables between groups

Variable Control group INOCA group p value

FCD (capillaries/mm²) 12.4 [11.2–15.8] 15.8 [13.1–17.9] 0.026

AFD (μm) 10.8 [9.1–14.1] 10.8 [7.9–11.7] 0.19

APD (μm) 14.1 [10.7–17.3] 13.9 [12.5–16.7] 0.68

EFD (μm) 15.9 [13.1–19.3] 14.2 [13.1–15.8] 0.075

RBCV (mm/s) 0.95 [0.94–0.97] 0.87 [0.85–0.92] 0.0001

RBCV_max (mm/s) 1.14 [1.12–1.16] 0.99 [0.95–1.02] 0.0001

TRBCV_max (s) 3.00 [3.00–4.00] 5.00 [4.00–7.00] 0.0004

Variable	Control group	INOCA group	p value
FCD (capillaries/mm²)	12.4 [11.2–15.8]	15.8 [13.1–17.9]	0.026
AFD (μm)	10.8 [9.1–14.1]	10.8 [7.9–11.7]	0.19
APD (μm)	14.1 [10.7–17.3]	13.9 [12.5–16.7]	0.68
EFD (μm)	15.9 [13.1–19.3]	14.2 [13.1–15.8]	0.075
RBCV (mm/s)	0.95 [0.94–0.97]	0.87 [0.85–0.92]	0.0001
RBCV_max (mm/s)	1.14 [1.12–1.16]	0.99 [0.95–1.02]	0.0001
TRBCV_max (s)	3.00 [3.00–4.00]	5.00 [4.00–7.00]	0.0004

Data are expressed as median [interquartile range]. INOCA Group - Ischemia with no obstructive coronary artery disease group. FCD - Functional Capillary Density. AFD - Afferent Capillary Diameter. APD - Apical Capillary Diameter. EFD - Efferent Capillary Diameter. RBCV - Red Blood Cell Velocity. RBCVmax - Maximal Red Blood Cell Velocity. TRBCVmax - Time to reach RBCVmax.

3.4 Endothelial injury/inflammatory biomarkers analysis

Intergroup differences regarding blood concentrations of endothelial injury/inflammatory biomarkers are presented on Fig. 1. Circulating levels of sVCAM-1 (p < 0.0001), ET-1 (p < 0.0001) and hsCRP (p = 0.003) were significantly higher in INOCA group in comparison to control one. No significant difference was observed in blood concentration between groups concerning sICAM-1 (p = 0.37), oxLDL (p = 0.62) and adiponectin (p = 0.99).

Fig. 1

Differences of endothelial injury/inflammatory biomarkers between control and INOCA groups (a-f) and Spearman correlations between maximal Red Blood Cell Velocity (RBCVmax) and relative increment of post-ischemic blood flow maximal related to baseline forearm blood flow (% Maximal post-ischemia blood flow) (g-k). Comparisons between control and INOCA groups concerning (a) Soluble Vascular Cell Adhesion Molecule (sVCAM-1), (b) Soluble Intercellular Adhesion Molecule (sICAM-1), (c) Endothelin-1 (ET-1), (d) Oxidized Low Density Lipoprotein (oxLDL), (e) Adiponectin and (f) High Sensitivity C-Reactive Protein (hs-CRP) concentrations. Data are expressed as median [interquartile range]. Spearman correlations between maximal Red Blood Cell Velocity (RBCVmax) and (g) sVCAM-1, (h) ET-1 and (i) hs-CRP in all groups. Spearman correlations for % Maximal post-ischemia blood flow and (j) sVCAM-1 and (k) ET-1 of all participants. One can note inverse correlations for all parameters evaluated.

3.5 Correlations between NVC and VOP variables and biomarkers concentrations

RBCV_max showed significant correlations with sVCAM-1 (r = –0.38, p < 0.05), ET-1(r = –0.73, p < 0.05) and CRP (r = –0.33, p < 0.05). Relative increment of maximal post-ischemic blood flow was significantly correlated to sVCAM-1 (r = –0.42, p < 0.05) and ET-1 (r = –0.48, p < 0.05), Fig. 1. No other significant correlations between VOP and NVC variables and biomarkers concentrations were found (Fig. 1).

4 Discussion

Reactive hyperemia is a well-recognized technique for noninvasive evaluation of peripheral microvascular function and an important predictor of cardiovascular morbidity and mortality [24]. In summary, reactive hyperemia reflects the magnitude of limb reperfusion in response to a period of blood flow interruption [24]. Reactive hyperemia after 5-minute forearm occlusion has been used in clinical studies to assess hyperemic flow velocity or volume flow by ultrasound, and hyperemic volume flow using VOP [25]. Human studies have also demonstrated an impairment of reactive hyperemia in patients with atherosclerosis [26, 27] and in patients with cardiovascular disease risk factors [28 –30]. The latter studies [29] and [30] also reported inverse correlations between hyperemic flow velocity and traditional coronary risk factors and systemic markers of inflammation. According to Rosenberry and Nelson [24] independently of the method used, impaired reactive hyperemia means (micro)vascular dysfunction.

Regarding VOP, INOCA group presented a significant impairment of microvascular function during the reactive hyperemia response, when compared to Control group (Table 3). Endothelium independent vasodilatation observed after nitroglycerin application was not significantly different between groups (Table 3), indicating that vascular wall function in INOCA group remained unaffected. Other study demonstrated that patients with microvascular angina had an impaired forearm hyperemic response, [13] which corroborates our findings.

The impaired microvascular function may be explained by the significantly greater plasma concentration of ET-1 in INOCA group compared to control group. As demonstrated on Fig. 1, relative increment of maximal post-ischemic blood flow was inversely correlated with ET-1 concentration (r = –0.48, p < 0.05). ET-1 increases VSMC tone, mitigating the effects of vasodilators released during the reactive hyperemia.

Our study has shown that INOCA group presents significantly lower RBCV and RBCV_max and longer TRBCV_max in comparison to control group (Table 4) demonstrating an impairment of microvascular function, more precisely on microvascular hemodynamics. Since microvascular dysfunction is a systemic phenomenon that occurs similarly in the entire body, [13] we believe that nailfold microvascular bed reflects what occurs in the coronary microcirculation of patients with INOCA. To our knowledge, this is the first time that impairment of microvascular hemodynamics in patients with INOCA was reported in the literature. It is possible that patients with INOCA possesses incipient atherosclerotic lesions which are unable to be detected during angiography but are sufficient to alter microvascular hemodynamics and being detected by NVC. The literature regarding NVC and advanced CAD is very scarce. One study demonstrated a significant reduction of FCD in early-onset CAD (EOCAD) in comparison to age and sex-matched healthy controls at basal conditions and during PORH using dorsal finger videocapillaroscopy (in which capillaries can be seen perpendicular to skin), suggesting that CAD in young adults is also accompanied by capillary rarefaction [31]. Using myocardial contrast echocardiography (MCE) was demonstrated that patients with CAD and left ventricular dysfunction with one or more coronary artery stenosis (≥70% diameter stenosis) also presented significant capillary rarefaction which was associated to a worse functional recovery following revascularization [32].

We did not observe significant differences on capillary morphology between groups (Table 4) in disagreement with Pasqui and co-workers [3]. Possibly, derangements in microvascular hemodynamics precede morphological changes in capillaries in patients with INOCA.

Some studies, have demonstrated that patients with chest pain and normal arteriograms, presented significantly lower FCD in skin when compared to controls [3, 33]. However, we found significantly higher FCD in INOCA group, which may be due to angiogenic stimulus elicited by chronic ischemia [34]. We found significantly elevated blood concentration of sVCAM-1 in INOCA group in comparison to control group (Fig. 1) that can be responsible for the angiogenic stimulus. sVCAM-1 is a classic biomarker of endothelial injury and, currently, it has been pointed out as an angiogenic factor being significantly greater in patients with stable angina and well-developed collateral coronary vessels [35].

A possible cause for microvascular dysfunction in patients with INOCA is the presence of significantly higher blood concentrations of CRP, an inflammatory and cardiovascular risk marker [36] In fact, we found a significant inverse correlation between RBCV_max and CRP (r = –0.33, p < 0.05), Fig. 1.

Factors that increase the inflammatory status (e.g. older age, elevated BMI, waist circumference and fat mass) were more prominent in INOCA group, when compared to the control group (Table 1), contributing for cardiovascular risk increase (Table 2) and, consequently, for higher circulating levels of CRP in these individuals (Fig. 1).

Several studies demonstrated that CMD in the presence or absence of obstructive CAD is an indicative of worse prognosis and with increased risk of major adverse cardiovascular events (MACE) [37], such as death, nonfatal myocardial infarction, nonfatal stroke, and heart failure hospitalization [38], In addition, reactive hyperemia has an independent association with cardiovascular outcomes [25] and is considered a predictor of all-cause of death and cardiovascular morbidity and mortality [24]. In this context, the evaluation of reactive hyperemia (using VOP or NVC techniques, for example) may constitute an important strategy to detect, noninvasively, peripheral microvascular dysfunction and to predict the incidence of cardiovascular events [39].

4.1 Limitations

INOCA group presented significantly greater mean age and BMI in comparison to control group. As we know, higher age and BMI may worse, per se, microvascular function and could affect the actual relationship between INOCA and peripheral microvascular function. For this reason, NVC and VOP variables were adjusted for age and BMI by ANCOVA to eliminate the influence of these confounding factors in data interpretation and conclusion.

In INOCA group, patients with arterial hypertension were treated by ACE inhibitors or angiotensin II receptor antagonists and patients with dyslipidemia were treated with statins. Even though ACE inhibitors, angiotensin II receptor antagonists and statins are drugs with recognized property to improve vascular function, INOCA group presented a significant impairment of microvascular function, which demonstrated that these drugs probably did not to mask the results.

5 Conclusion

Patients with ischemia and no obstructive coronary artery disease (INOCA) present an impairment of microvascular function and hemodynamics that could be detected in the peripheral circulation and may reflect what occurs in the coronary microcirculation, reinforcing the hypothesis that microvascular dysfunction is a systemic condition.

Funding

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Conflicts of interest

The authors declare that there is no conflict of interest.

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