Capillary microvascular function in patients with liver cirrhosis: a nailfold video-capillaroscopy study

Introduction

Liver cirrhosis represents a major public health issue and a significant factor in morbidity and mortality worldwide [1,2]. Since its early stages are asymptomatic, the prevalence of cirrhosis is underestimated; population-based studies reported a prevalence of 0.3%, but autopsy studies from the general population have found rates up to 4.5-9.5% [1,3,4]. In developed countries, the main causes of cirrhosis are viral hepatitis and misuse of alcohol, while more recently, non-alcoholic liver disease, an entity strongly associated with cardiovascular disease, is increasingly recognized as a major cause of cirrhosis [1,5].

Endothelial dysfunction refers to a state of imbalance between vasodilating and vasoconstricting factors, inappropriately favoring the latter [6]. Nitric oxide (NO) is directly associated with endothelial function, as decreased bioavailability of NO is the hallmark of endothelial dysfunction [7]. In liver cirrhosis, a major dysregulation of the circulatory systems is observed, characterized by alterations in both intrahepatic and systematic vasculature; these are associated with imbalance of vasoactive agents, including mainly NO [8]. In particular, reduced NO bioavailability, oxidative stress, and endothelial dysfunction in the intrahepatic circulation result in significant intrahepatic vasoconstriction and increased vascular resistance, leading to portal hypertension [8]. On the other hand, NO overproduction is observed in systematic vascular beds, causing arterial vasodilation and hyperdynamic circulatory syndrome [8]. Although alterations in the intrahepatic circulation and the systematic microcirculation have both been well described, the peripheral microcirculation in cirrhosis is less well studied. There are a few studies describing peripheral microvascular alterations in the literature, but with conflicting results [9,10]. Armentano et al, in a small pilot study, found that flow-mediated dilatation (FMD) was higher in Child-Pugh C than Child-Pugh B and Child-Pugh A, suggesting an increased peripheral vasodilatory response with the progression of the disease [11]. Similarly, Berzigotti et al indicated that FMD increased in parallel with Child-Pugh score [12]. On the other hand, Ponziani et al found that FMD was lower in patients with cirrhosis than in healthy controls, while Marcacci et al suggested that FMD, and consequently peripheral endothelial function, was reduced in worsening cirrhosis [13,14].

During the last decades, several methods have been developed to assess endothelial and microvascular function in humans [9,15]. Nailfold capillaroscopy is a noninvasive method that provides a detailed examination of the number and the morphology of the nailfold capillaries, giving valuable information about microcirculatory derangement [9]. Nailfold video-capillaroscopy (NVC), a development of the aforementioned method, offers the opportunity to evaluate capillaries’ functional alterations—and consequently microvascular function—by measuring capillary density during different vascular phases (baseline, post-arterial occlusion and after venous congestion) [16]. NVC has been used to assess peripheral microcirculation function in patients with hypertension, diabetes mellitus, rheumatoid arthritis and chronic kidney disease [16-19]. To the best of our knowledge, no study has examined microvascular endothelial function in individuals with cirrhosis using NVC and employing the standard methodology.

The aim of the present study was to evaluate microvascular function with the use of NVC in patients with cirrhosis vs. controls, as well as at different stages of cirrhosis by evaluating differences between the various Child-Pugh classes. As secondary objective, we examined possible associations between microvascular function and markers of macrovascular function, including parameters such pulse wave velocity (PWV), wave reflection indices and carotid intima–media thickness (cIMT).

Patients and methods

NVC

NVC was performed using a OptiPix Capillaroscopy Clinic 1.7.x device (magnification 200×). Using digital video, capillaries were depicted on the dorsal skin of the distal phalanx of the third and fourth fingers of the right hand. The imaging was performed in the middle of the nailfold, ~4.5 mm from the terminal capillary line. The researcher chose a visual field of 1 mm² and the number of capillaries was counted offline from a freeze-framed reproduction of the video. The capillary density was measured (as the mean of the 2 fingers) in 3 different phases, as previously described in the literature (Fig. 1) [16,22]. Baseline capillary density was measured during a period of 15 sec, and only continuously erythrocyte-perfused capillaries were counted. Subsequently, arterial occlusion was applied, using a small cuff at the base of the studied finger that was inflated to 260 mmHg for 4 min. Capillary density during post-occlusive reactive hyperemia was defined as the number of all (continuously and intermittently perfused) capillaries that were counted during 15 sec immediately after the release of the cuff. Fifteen min following the post-occlusive reactive hyperemia phase, venous congestion was induced by inflating the cuff to 60 mmHg for 2 min. Immediately after that, all (continuously and intermittently perfused) capillaries were counted for 15 sec and the capillary density after venous congestion was recorded. In addition, the absolute increase in capillary density from baseline to the post-occlusive reactive hyperemia phase was calculated by subtracting the baseline capillary density from the post-occlusive reactive hyperemia capillary density. The % capillary recruitment was also determined by dividing the absolute increase in capillary density in post-occlusive reactive hyperemia by baseline capillary density and multiplying the result by 100.

Figure 1 Images of (A) the nailfold video-capillaroscopy device, and capillary density in a patient with cirrhosis at (B) baseline, (C) post-occlusive hyperemia, and (D) venous congestion

Results

Participant characteristics

Fig. 2 presents the study flowchart. A total of 80 participants were included in the study: 60 patients with cirrhosis (20 in each group for Child-Pugh classes A, B and C) and 20 controls. Demographic, clinical and laboratory characteristics of the study population are presented in Table 1. For patients with cirrhosis, the primary liver disease was alcohol-related liver disease in 28 patients (46.7%), metabolic dysfunction-associated steatotic liver disease in 13 patients (21.7%), autoimmune hepatitis in 9 patients (15%), chronic hepatitis B in 8 patients (13.3%), and unknown/cryptogenic in 2 patients (3.3%). Twenty patients (33.3%) had compensated cirrhosis and 40 decompensated cirrhosis (66.7%). There were no significant differences between groups in age, sex or body mass index. As expected, model for end-stage liver disease (MELD) score and MELD-Na score were progressively larger, while platelet count, hemoglobin levels and albumin levels were lower in more advanced Child-Pugh classes.

Figure 2 Study flowchart

Table 1 Demographic, anthropometric and clinical characteristics of the study participants

NVC parameters in cirrhotic patients and controls

Table 2 presents the differences in NVC parameters between patients with cirrhosis and controls. Baseline capillary density was lower in patients with cirrhosis than in controls (35.8±3.6 vs. 38±1.1 capillaries/mm², P=0.01). Similarly, cirrhotic patients presented significantly lower capillary density both during post-occlusive reactive hyperemia (40.0±4.4 vs. 45.3±1.5 capillaries/mm², P<0.001) and after venous congestion (43.3±4.2 vs. 47.2±1.5 capillaries/mm², P<0.001). Capillary recruitment was significantly lower in patients with cirrhosis compared to controls (11.8±4.6% vs. 19.1±2.6%, P<0.001).

Table 2 Nailfold video-capillaroscopy parameters in patients with cirrhosis and controls

NVC parameters in different Child-Pugh classes

Fig. 3 presents the differences in NVC parameters between patients with different Child-Pugh classes. Capillary density in all phases (baseline, during post-occlusive reactive hyperemia and after venous congestion) decreased significantly with deterioration of Child-Pugh class (Fig. 3). Similarly, capillary recruitment became significantly lower as the Child-Pugh class increased (controls: 19.1±2.6%, Child-Pugh A: 15.1±5.1%, Child-Pugh B: 11.9±2.9, Child-Pugh C: 8.3±2.7%, P<0.001).

Figure 3 Capillary density at baseline (A), during post-occlusive reactive hyperemia (B), after venous congestion (C) and capillary recruitment (D) in different study subgroups

Arterial stiffness, wave reflections and cIMT in cirrhotic patients and controls, and in different Child-Pugh classes

Supplementary Table 1 presents the differences in central blood pressure, wave reflection parameters, arterial stiffness and cIMT between cirrhotic patients and controls. No significant differences were detected in aortic SBP or DBP pressure levels between the 2 groups. Similarly, wave reflection parameters (AP, AIx, AIx75), PWV and cIMT were similar between the groups.

Aortic SBP and aortic DBP decreased with deterioration of Child-Pugh class (Child-Pugh A: 122.4±16.8/79.6±12.6 mmHg, Child-Pugh B: 118.2±12.9/73.4±9.8 mmHg, Child-Pugh C: 104.7±12.2/68.7±6.1 mmHg, P=0.001 and P=0.002, respectively). No significant differences were detected in wave reflections, PWV or cIMT among different Child-Pugh classes (Table 3).

Table 3 Arterial stiffness, wave reflection parameters and cIMT in the different study sub-groups

Discussion

This study used NVC to compare microvascular function between patients with cirrhosis and controls, as well as among different stages of cirrhosis, and evaluated capillary density at rest, during post-occlusive reactive hyperemia and after venous congestion. Capillary density was significantly lower in cirrhotic patients compared to controls in all phases of NVC. Moreover, capillary density decreased with the deterioration of Child-Pugh stages. Similarly, capillary recruitment became progressively lower with worsening stages of cirrhosis, suggesting that increasing severity of cirrhosis is associated with more impaired peripheral microcirculatory function. This observation was also reinforced by the fact that capillary density and capillary recruitment were inversely correlated with MELD/MELD-Na score, total bilirubin and INR, whereas they were positively associated with albumin.

The endothelium is the inner cellular lining of blood vessels, which serves an important role in many vascular functions [26]. It is critically involved in the control of vasomotor tone and vascular permeability, procoagulant and anticoagulant activity, angiogenesis, inflammatory processes, and innate and acquired immunity [26]. Endothelial dysfunction refers to a state of imbalance between vasodilating and vasoconstricting factors, inappropriately favoring the latter [6]. It is considered as an early sign of microcirculation disruption and is associated with adverse cardiovascular outcomes [27-29]. Over the years, several methods have been used to assess peripheral endothelial and microvascular function in human studies, including venous occlusion plethysmography, flow-mediated dilatation (FMD), laser Doppler flowmetry, laser-speckle contrast imaging analysis and NVC [9]. The aforementioned methods have also been used in patients with cirrhosis with conflicting results [9,10]. A few studies found that FMD was lower in the early stages of cirrhosis, indicating a heightened vasodilatory response with the progression of the disease [11,12,30]. On the other hand, a number of studies have shown that peripheral endothelial function deteriorates progressively towards the advanced stages of cirrhosis [13,14,31]. In a recent meta-analysis by our group, we found no difference in peripheral endothelial function, evaluated with semi-invasive or noninvasive methods, between patients with cirrhosis and controls, suggesting that the heterogeneous design of the included studies and potential confounding variables, such as the presence of cardiovascular risk factors, could have contributed to the above result [10].

NVC is a simple, noninvasive, dynamic method that can be used to evaluate endothelial function and microcirculation [16]. Capillary rarefaction refers to the reduction of capillary density in any given visual field, and can be divided into structural/anatomical, defined as a decrease in the overall number of capillaries, and functional, defined as a reduction in the number of perfused capillaries [32]. NVC, using post-occlusive reactive hyperemia and venous congestion, can provide information on both types of capillary rarefaction, offering important advantages compared to simple capillaroscopy. In particular, capillary density after venous congestion is considered to objectively determine the anatomic capillary number, as it can reveal non-perfused capillaries that may be overlooked through simple capillaroscopy [16]. Additionally, capillary density during post-occlusive reactive hyperemia evaluates the functional recruitment of initially non-perfused capillaries, being an indicator of both structural and functional integrity [16]. To the best of our knowledge, only 1 study has used NVC in patients with cirrhosis [33]. Brito-Azevedo et al used NVC to evaluate the microcirculation in cirrhotic patients and compare it with inflammatory parameters [33]. However, the authors did not apply the standardized methodology, as previously described in the literature, to assess the microcirculatory function, and they did not provide information about capillary density during post-occlusive reactive hyperemia and after venous congestion, which could be compared with our results [33].

Our study had a different scope: to evaluate peripheral microvascular function and to examine potential correlations with macrovascular parameters, in order to achieve a more accurate approach to changes in the peripheral circulation in patients with cirrhosis. First, we showed that baseline capillary rarefaction is greater in cirrhotic patients than in controls, and deteriorates with progressing Child-Pugh stages. In addition, patients with cirrhosis exhibited lower capillary density than controls, both during post-occlusive reactive hyperemia and after venous congestion, a difference that becomes progressively worse as the disease deteriorates, suggesting that cirrhosis impairs both structural and functional capillary integrity, while the severity of the disease exacerbates this impairment.

NVC has already been implemented in the evaluation of patients with various conditions, mainly patients with rheumatologic disorders; however, an increasing body of research now extends its application to hypertension, diabetes mellitus and chronic kidney disease [16-19]. Based on our findings, this technique could also be valuable in cirrhosis, potentially identifying patients with more advanced clinical status, impaired peripheral microcirculation and a worse prognosis. In this context, and given the simple, noninvasive nature of the methodology, NVC may represent a promising tool for bedside risk stratification. Furthermore, integrating NVC into routine hepatology practice could enable noninvasive monitoring of microvascular health over time, providing additional information beyond conventional biochemical and hemodynamic parameters. Serial NVC assessments might, for example, help track the progression of microvascular dysfunction, or evaluate responses to therapeutic interventions. Nevertheless, evidence in this area remains limited, and further research is needed to standardize protocols, validate prognostic thresholds, and determine the feasibility of incorporating this methodology into everyday clinical practice.

Regarding the macrocirculation, PWV, the gold-standard measure of arterial stiffness [34], and cIMT, a common measure of atherosclerosis [35], we found no significant differences between cirrhotic patients and controls, or between different stages of cirrhosis. Our findings are in partial agreement with existing studies. Novo et al found that PWV was significantly higher in patients with hepatitis C virus (HCV) cirrhosis compared to controls, suggesting that HCV leads to subclinical vascular damage [36]. Huang et al measured PWV and demonstrated that cirrhotic patients had higher values than controls, while they found no disparities among different etiologies of cirrhosis, or between compensated and decompensated disease [37]. The absence of such differences (between patients and controls) in our study could probably be associated with the sample size. However, we demonstrated a mild reverse correlation between cIMT and capillary density, both during post-occlusive reactive hyperemia and after venous congestion, suggesting that microcirculatory dysfunction may be associated with macrovascular impairment in cirrhotic patients.

This study has several strengths. It is the first to examine the full spectrum of peripheral circulation alterations in patients with different stages of cirrhosis, including valid methods for evaluating both microcirculation (NVC) and macrocirculation (arterial stiffness, cIMT). We applied a strict and detailed protocol to assess microcirculation with NVC, adhering to the standardized methodology in the literature. The main limitation of the study is its observational character, which prevents the establishment of cause-and-effect relations. Although this is the largest study in the field, the sample was insufficient for conducting subgroup analyses that would have enabled us to examine the influence of additional factors on the differences we demonstrated. Finally, our findings regarding microvascular function should be interpreted with caution when extrapolating to other vascular beds. Endothelial cells throughout the vascular tree display considerable phenotypic heterogeneity in both structure and function; therefore, observations derived from one arterial or microvascular bed cannot be directly generalized to others [26]. Combining NVC with additional noninvasive techniques, such as the assessment of skin microvascular function using laser Doppler flowmetry and intradermal microdialysis, may offer a more comprehensive characterization of the peripheral microcirculation in patients with cirrhosis. While NVC provides high-resolution structural information on capillary density and recruitment, laser Doppler flowmetry can dynamically quantify cutaneous blood flow responses, while intradermal microdialysis allows a localized assessment of the release of endothelial mediators, such as nitric oxide or acetylcholine-induced vasodilation [9]. Future studies may therefore benefit from protocols that incorporate more than 1 complementary technique.

In conclusion, this study showed that capillary density at rest, during post-occlusive reactive hyperemia and after venous congestion, was lower in cirrhotic patients compared to controls, while it further deteriorated with advanced disease, as indicated by progressive Child-Pugh stages, suggesting that cirrhosis is characterized by impaired peripheral microvascular function, and that disease severity contributes to further microcirculation dysfunction. As endothelial dysfunction and microcirculatory disturbances are associated with adverse outcomes in different populations, further studies are needed to explore the potential effects of these findings on morbidity and mortality in patients with cirrhosis.