Evaluation of atherosclerotic risk in rosacea patients through serum fetuin‐A and carotid intima media thickness

The link between rosacea and various systemic conditions has been growing in prominence, even though the relationship between rosacea and cardiovascular disease remains a subject of debate in current research.


| INTRODUC TI ON
Rosacea is a cutaneous inflammatory disease.Persistent centrofacial erythema, flushing, telangiectasia, papules, pustules, less frequently phymatous changes, and eye involvement are the characteristics of the disease.There is insufficient evidence about the etiopathogenesis of the disease.4][5][6] Rosacea and CVD share some common immune and cellular activation pathways.Some mediators such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-17, reactive oxygen species (ROS), and matrix metalloproteinases (MMP) are produced as a result of these pathways. 7,8e presence of various systemic comorbidities, along with the existence of similar underlying immune mechanisms, and the detection of increased C-reactive protein (CRP) and oxidative mediators in rosacea patients, suggest the presence of underlying low-grade systemic inflammation in rosacea, similar to what is observed in psoriasis. 7Inflammation has a significant impact in the initiation and progression of atherosclerotic process by causing endothelial dysfunction. 9rdiovascular calcification is considered a marker of advanced stage of atherosclerotic disease and has been associated with three to four times increase in cardiovascular morbidity and mortality risk. 10Fetuin-A (FA) and matrix gla protein (MGP) are biomarkers of cardiovascular calcification. 11,12rly detection of comorbidities, including the early stages of CVD, is crucial for effective management and prevention of adverse outcomes.Using easily measurable biomarkers, such as proteins or glycoproteins, that are associated with an increased cardiovascular risk can be invaluable in this regard.For this purpose, we aim to investigate the levels of hs-CRP, FA, and MGP and to assess the carotid intima media thickness (CIMT) using ultrasonography, which serve as marker of atherosclerosis and cardiovascular calcification, in rosacea patients.We also intend to explore their correlation to the disease subtype and severity.

| Participants
A total of 50 rosacea patients (37 female, 13 male) and 49 controls (36 female, 13 male) has been enrolled in this prospective, crosssectional study.The control group was selected from healthy employees in our hospital using snowball technique.Age < 18 years, pregnancy, lactation, concomitant active or chronic infection, DM, cerebrovascular disease, coronary artery disease (CAD), chronic renal or hepatic disease, other chronic inflammatory or autoimmune disease, and consumption of any medication for rosacea in last 3 months were exclusion criteria.
Dermatologic examination was conducted by same dermatologists.Rosacea patients were classified into four subtypes based on the classification defined by the National Rosacea Society. 13tzpatrick skin type, age of onset, disease duration (years), and localization were recorded.The rosacea clinical scoring system (Rosacea Clinical Scorecard) was used to determine the severity of disease, in which signs and symptoms are graded on a scale of 0-3 as none, mild, moderate, or severe.This results in the classification of rosacea severity score as mild (0-9), moderate (10-18), and severe (≥19). 14,15rticipants' lifestyle factors were documented.Smokers were categorized as current, ex or non-smoker.Alcohol consumption was assessed, with regular consumption defined as ≥3 times per week.
Additionally, participation in regular physical activity was recorded, involving exercise sessions lasting ≥45 min, at least three times weekly.Anthropometric measurements were conducted, encompassing weight (kg), height (cm), and waist circumference (WC) (cm).
Weight/height 2 (kg/m 2 ) was the formula of body mass index (BMI).
Blood pressure was measured using sphygmomanometer (ERKA, The Original, VARIO, Germany) after a 10-min rest period.The mean of right and left arm values was considered as the individual's systolic (SBP) and diastolic blood pressure (DBP).

| Laboratory analysis
Venous blood samples were taken from all subjects after a 12-h fasting, and centrifugated at 4000 rpm for 10 min at +4°C.Samples were aliquoted and stored at −80°C until the day of analysis.Serum hs-CRP was determined by the turbidometric method using commercial kits on the Cobas® 8000 modular auto-analyzer (Roche Diagnostics GmbH, Mannheim, Germany).Serum total cholesterol, triglyceride, and high density lipoprotein (HDL) levels were analyzed using the enzymatic colorimetric method on the Cobas-8000 device (Roche Diagnostics GmbH, Mannheim, Germany).Serum low density lipoprotein (LDL) levels were calculated using the Friedewald formula

| Ultrasonographic evaluation
The CIMT was measured using B-mode equipment (Toshiba Aplio 500, Toshiba Medical Systems, Co. Ltd., Otawara, Japan) with a 6-8-MHz high resolution linear probe with a 35 head extension by an experienced radiologist blinded to the participants.The common carotid artery was scanned longitudinally in supine position.Measurements were performed bilaterally from three contiguous sites at 5 mm intervals in each side at a distance of 10 mm proximal to the carotid artery bifurcation, and CIMT consisted of the average of the three measurements.The "mean CIMT" value is obtained by calculating the average of the right and left CIMT, and values greater than 75th percentile specific for age group and gender were considered high and indicative of subclinical atherosclerosis. 16

| Statistical analysis
The statistical analyses were carried out utilizing SPSS software version 23.To ascertain the normal distribution of variables, the Shapiro-Wilk test was employed.Descriptive analyses were presented as mean ± standard deviation for normally distributed variables, and as median (min-max) for non-normally distributed and ordinal variables.Categorical variables were presented as frequency and percentage.For comparing differences in categorical variables between the patient and control groups, the chi-squared test or Fisher's exact test (when chi-squared test assumptions were not met due to low expected cell counts) were used.The Mann-Whitney U test was utilized to compare non-normally distributed numeric and ordinal variables between the patients and the control.Additionally, the independent samples t-test was used to compare normally distributed numeric variables.Pearson or Spearman's correlation analysis was conducted to assess associations between the study parameters.A p < 0.05 was considered statistically significant.

| RE SULTS
In the study, a total of 50 patients (37 female and 13 male) with a mean age of 41.74 ± 13.15 years and 49 control (36 female and 13 male) with a mean age of 40.29 ± 12.5 years were included.No significant differences were observed between the two groups regarding mean age and sex (p = 0.732, p = 0.952, respectively).The rosacea subtypes were distributed as follows: 24 patients exhibited the erythematotelangiectatic (ETR), 24 patients had the papulopustular (PPR), and 2 patients presented with the phymatous subtype.The mean duration of the rosacea was 5.9 years.Twenty-five patients had mild, 23 patients had moderate, and 2 patients had severe rosacea according to Rosacea Clinical Scorecard.Ten (20%) of patients had eye involvement, 21 (42%) had positive family history of rosacea and 30 (60%) had positive family history of CVD.
The rosacea group exhibited a significantly higher level of hs-CRP (p = 0.009), while the serum level of FA was significantly lower in comparison to the control (p < 0.001).Additionally, the mean CIMT (p = 0.001) was significantly greater and the number of patients with mean CIMT>75th percentile according to age and gender (p = 0.001) were significantly higher in the rosacea group.No significant difference was found between the patients and the control in terms of smoking status, alcohol consumption, regular physical activity, family history of CVD, BMI, WC, SBP, DBP, MGP, and serum lipid parameters.The descriptive characteristics are presented in Table 1.
Given the limited number of patients with the phymatous subtype (only two), they were excluded from the analysis.When patients with ETR and PPR subtypes were compared, no significant differences were observed between these two subtypes in terms of BMI, WC, SBP, DBP, serum lipid parameters, hs-CRP, FA, MGP, and mean CIMT (Table 2).
When conducting analyses based on disease severity, moderate and severe patients were combined into a single group called "moderate-severe" and compared with mild group.Patients within the moderate-severe disease category exhibited a significantly higher BMI and WC compared to those with mild disease (p = 0.006 and p = 0.043, respectively).Systolic blood pressure, DBP, serum lipid parameters, hs-CRP, FA, MGP, and mean CIMT were not statistically different between groups (Table 2).
No significant difference was found between these groups in terms of other study parameters.
Within the rosacea group, a moderate correlation was observed between disease severity and BMI (r = 0.4, p = 0.004), while a weak positive correlation existed between disease severity and WC (r = 0.288, p = 0.043).In contrast, no significant associations were noted between the study parameters and the duration of the disease.Further details regarding the correlation between the severity and duration of rosacea and the study parameters are provided in Table 3.

| DISCUSS ION
Rosacea is a skin disease affecting predominantly the central face.
[19][20][21][22][23] The underlying mechanisms of the relationship between rosacea and systemic diseases are not fully understood.Chronic inflammation, which arises from the activation of mediators and cells involved in innate and adaptive immunity, neurogenic inflammation, environmental, and genetic factors are believed to play a role in this association. 7,8Furthermore, multiple studies have indicated that systemic markers of inflammation, such as CRP, ROS, IL-8, IL-1β, and TNF-α, are elevated in individuals with rosacea compared to those without it.Drawing parallels with conditions like psoriasis, this suggests that inflammation in rosacea extends beyond the skin, hinting at the presence of systemic inflammation linked to the ailment. 24,25A B L E 1 Descriptive data of patients and the control group.Th17 cells, B cells, and the vascular endothelium. 7,83][4][5][6] A retrospective cross-sectional study, involving 33 553 rosacea patients, elicited an increased risk of concurrent CAD and rosacea after adjusting for specific CVD risk factors.In most cases, it was observed that CVD began before the development of rosacea. 4Similarly, a study indicated that individuals with rosacea exhibited a higher prevalence of hypercholesterolemia, hyperlipidemia, elevated CRP levels, family history of CVD, smoking, and heavy alcohol consumption when compared to control subjects. 3A single-center study highlighted a connection between the severity of rosacea and an elevated risk of associated conditions such as hyperlipidemia, hypertension, and CVD. 23In a substantial Danish study (n = 35 958), there was no discernible increase in the risk of death related to CVD when examining cause-specific mortality among rosacea patients. 5In a review published in 2018, rosacea showed a significant association with CVD risk factors, encompassing CAD, peripheral artery disease, heart failure, DM, hypertension, dyslipidemia, and metabolic syndrome.Notably, even after adjusting for hypertension, DM, and dyslipidemia, the link between rosacea and CAD remained statistically significant. 2In a meta-analysis involving 50 442 rosacea patients, conducted by Chen and colleagues indicated that individuals with rosacea showed a higher prevalence of dyslipidemia, hypertension, elevated total cholesterol, increased levels of LDL, raised triglycerides, elevated SBP and DBP, and higher fasting blood glucose in comparison with those without rosacea.However, the study did not establish any connections between rosacea and ischemic heart disease, stroke, DM, or HDL. 6A recent systematic review and meta-analysis, which included 11 published studies, reported that rosacea is associated with high insulin resistance or DM, high SBP, dyslipidemia, and CVD. 26In another recent systematic review and meta-analysis, which evaluated 20 studies involving 40 752 patients, Tsai and colleagues reported that SBP, DBP, total cholesterol, LDL, CRP, and EFT values were greater, and the incidence of hypertension and insulin resistance was higher in rosacea patients compared to control.Based on these results, researchers have suggested that rosacea patients tend to show an increased subclinical cardiovascular risk. 27ronic inflammatory diseases such as psoriasis and rosacea are believed to initiate and advance the atherosclerotic process, which is an important cause of death, by inducing endothelial dysfunction. 9In this study, we found that in patients with rosacea, hs-CRP and mean CIMT values were significantly higher, while serum FA values were significantly lower compared to the control group.
In a cross-sectional study, rosacea patients exhibited significantly greater epicardial fat thickness (EFT) and mean CIMT measurements than the control (p < 0.001).In this study, multivariate logistic regression analysis revealed that EFT was independently associated with the presence of rosacea. 28In an additional case control study comparing 52 rosacea patients with 52 healthy controls, the patients had significantly higher EFT (p < 0.001), CIMT (p < 0.001), CRP (p = 0.004), total cholesterol (p = 0.003), and LDL (p = 0.004) levels.Differing from our study, total cholesterol and LDL levels, each being cardiovascular risk factors, were higher in the rosacea patients compared to the control.However, it was noted that total cholesterol, EFT, and CIMT were independently associated with rosacea, and EFT and CIMT were suggested to be simple and cost-effective measurements that can be used to determine subclinical cardiovascular risk in rosacea. 29In a case-control study that assessed subclinical atherosclerosis and cardiovascular risk using EFT and ankle-brachial index (ABI), it was found that the mean EFT was greater in patients with rosacea (p < 0.001).The authors suggested that subclinical cardiovascular risk might be increased in rosacea, recommending that rosacea patients should be monitored in this regard. 30In another recent study comparing 44 patients with rosacea and 44 controls, the findings revealed that serum IL-1β (p < 0.001), IL-6 (p < 0.001), TNF-α (p < 0.001), and hs-CRP (p < 0.001) levels were notably elevated in patients with rosacea.However, the mean CIMT values did not display a significant difference between groups.The researchers have indicated that this outcome could be attributed to the exclusion of patients with conventional cardiovascular risk factors from the study.Based on their own findings, the researchers have argued that rosacea may not be an independent risk factor for the development of atherosclerosis. 25In our study, there were no differences between as a biomarker of CVD. 31 In the literature, there are various casecontrol studies that have investigated CRP levels in rosacea patients.Across all of these studies, a consistent pattern has emerged, demonstrating significantly elevated CRP levels in rosacea patients as compared to the control group. 3,29Similar to our study, a separate study evaluating hs-CRP levels alongside other proinflammatory cytokines reported significantly elevated hs-CRP levels in rosacea patients relative to controls. 25tuin-A is a multifunctional glycoprotein which has inhibitory effects on vascular calcification and coronary artery calcification (CAC), mineralization, kidney stone formation, systemic inflammation, and chronic tissue damage.It functions as a potent anti-inflammatory cytokine, participating in macrophage deactivation, the prevention of calcification in vascular smooth muscle cells, and functioning as an antagonist to transforming growth factor (TGF)-β. 32 Numerous studies have indicated that low serum levels of FA is an independent risk factor for CVD, and these levels can be employed to predict the likelihood of developing CAC. 11,33ere is no study available in the English literature that assesses serum FA levels in rosacea patients.In this study we found a significant reduction in FA levels among rosacea patients when compared to the control.This finding lends support to the notion that there could be an elevated risk of CAC and increased atherosclerosis in individuals with rosacea.
Matrix gla protein, a vitamin K-dependent protein which is secreted primarily by vascular smooth muscle cells in the arteries, is a potent inhibitor of vascular calcification. 34Several studies have demonstrated that the level of MGP increases in individuals with acute coronary diseases. 12,34Contrary to a unified conclusion, the existing literature presents divergent findings regarding the relationship between MGP levels and vascular calcification.correlation between MGP levels and the extent of CAC.Additionally, variations in MGP fractions, including phosphorylated, unphosphorylated, carboxylated, and undercarboxylated forms, have yielded disparate outcomes. 34In our novel study, the first to assess MGP levels in rosacea patients, we identified no significant disparity in MGP levels between rosacea patients and the control.We think that evaluating the defined subfractions of the MGP could yield different results.
In our study patients within the moderate-severe disease category exhibited a significantly higher BMI and WC compared to those with mild disease.Also a positive correlation was found between the disease severity and these anthropometric measurements.It has been reported in various publications that obesity is one of the comorbidities associated with rosacea. 2,7,23Furthermore, it has been indicated that the risk of obesity is higher in individuals with moderate to severe rosacea compared to those with mild rosacea. 23In this regard, our findings are consistent with the literature.On the other hand, hs-CRP, FA, MGP, and mean CIMT were not statistically different between mild and moderate-severe groups in our study.Unlike our study, Ertekin et al.'s study found that individuals with moderate to severe rosacea had significantly higher CIMT values compared to those with mild rosacea. 25This variation in outcomes could potentially be attributed to differences in the studied populations, methodologies, or other contributing factors.
In our study, it was found that patients with ocular involvement had higher hs-CRP levels compared to those without involvement.
In ocular rosacea, similar to cutaneous involvement, it is known that the activation of both the innate and acquired immune systems and vascular dysregulation play a role in the pathogenesis.Rosacea patients demonstrate elevated levels of IL-1α, IL-1β, IL-16, TNF-α, monocyte chemoattractant protein-1, MMP-8, and MMP-9 in their tears. 35In Ertekin et al.'s study, there was no significant differences between rosacea patients and controls in terms of hs-CRP and other proinflammatory cytokine levels.However, in this study, CIMT levels were found to be higher in patients with ocular involvement. 25ese results raise the possibility of increased subclinical systemic

CO N FLI C T O F I NTE R E S T S TATE M E NT
Authors declare no conflict of interests for this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been re-

F I G U R E 1
The comparison of hsCRP values between rosacea patients with and without eye involvement.rosacea patients and the control group in terms of traditional cardiovascular risk factors, such as age, gender, BMI, WC, smoking-alcohol use, exercise status, SBP, DBP, total cholesterol, LDL, HDL, and trigliseride levels.Notably, our study revealed not only significantly higher mean CIMT values in rosacea patients compared to controls, but also a substantially greater risk of subclinical atherosclerosis, as indicated by age-and gender-specific CIMT values and percentiles.Furthermore, considering the heightened hs-CRP levels and the reduced serum FA levels in rosacea patients versus controls-both recognized as atherosclerotic risk factors-we contend that rosacea stands as an independent risk factor in the progression of atherosclerosis.C-reactive protein is widely employed as an indicator of inflammation and tissue damage.High-sensitivity assays for CRP yield hs-CRP, which is recognized by numerous authors as an excellent inflammation marker.The utilization of hs-CRP has revealed low-grade and subclinical systemic inflammation across various conditions, including CVD and DM.Furthermore, increased levels of hs-CRP are seen in various stages of atherosclerosis, and it has been introduced While certain studies have failed to establish a connection between MGP levels and vascular calcification, others have suggested an inverse TA B L E 3 Correlation between the severity and duration of rosacea and study parameters.
inflammation and CVD risk in patients with ocular involvement compared to those without.Further randomized controlled trials with larger numbers of patients are needed to investigate this relationship more thoroughly.The prospective and controlled nature of the study, the similarity of the patient and control groups in terms of traditional cardiovascular risk factors, and the evaluation of CIMT based on percentiles determined by age and gender, and the fact that FA, an important atherosclerotic risk factor, has been studied for the first time in patients with rosacea are the main strengths of our study.One of the limitations of the study is its single-center nature which could impact the generalizability of the findings to a broader population.The limited number of cases, especially the scarcity of patients with severe rosacea, is another restrictive aspect of the study.Another limitation is the lack of assessment of other subclinical atherosclerosis markers like EFT, flow-mediated dilatation, and ABI.These additional measurements could have provided a more comprehensive evaluation of subclinical atherosclerosis and its potential association with rosacea.In conclusion, this study demonstrated that rosacea patients had significantly higher levels of hs-CRP and mean CIMT, and significantly lower levels of serum FA, which are subclinical atherosclerosis indicators, compared to healthy controls.Furthermore, the assessment of CIMT based on age-and gender-specific percentile values unveiled a heightened subclinical atherosclerosis risk in rosacea patients relative to controls.Importantly, the levels of these inflammatory markers and CIMT showed no significant variation based on rosacea severity, duration, or clinical subtype.Considering these findings, irrespective of the severity, duration, or subtype of rosacea, we assert that it stands as an independent risk factor for the development of subclinical atherosclerosis.Consequently, individuals diagnosed with rosacea should undergo evaluation and vigilant monitoring for potential cardiovascular complications.Additionally, our study indicates a potential heightened risk of subclinical inflammation in patients with ocular involvement compared to those without.Extensive further studies are necessary to gain a comprehensive understanding of this matter.AUTH O R CO NTR I B UTI O N S Conceptualization, Sevgi Kulaklı and Sembol Yıldırmak; Data curation, Sevgi Kulaklı, Işıl Deniz Oğuz and Murat Usta; Formal analysis, Sevgi Kulaklı, Işıl Deniz Oğuz, Murat Usta and Alptekin Tosun; Funding acquisition, Sevgi Kulaklı and Sembol Yıldırmak; Investigation, Sevgi Kulaklı, Sembol Yıldırmak, Işıl Deniz Oğuz, Murat Usta, Alptekin Tosun and Burak Akşan; Methodology, Sevgi Kulaklı and Sembol Yıldırmak; Project administration, Sevgi Kulaklı, Sembol Yıldırmak, Işıl Deniz Oğuz, Murat Usta, Alptekin Tosun and Burak Akşan; Resources, Sevgi Kulaklı; Software, Sevgi Kulaklı and Işıl Deniz Oğuz; Supervision, Sembol Yıldırmak; Validation, Sevgi Kulaklı and Sembol Yıldırmak; Visualization, Sembol Yıldırmak, Murat Usta and Burak Akşan; Writing -original draft, Sevgi Kulaklı; Writingreview & editing, Sembol Yıldırmak and Işıl Deniz Oğuz.FU N D I N G I N FO R M ATI O N This research was funded by Scientific Project Office of Giresun University (Project number: SAĞ-BAP-A-250221-33).
ceived.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Clinical Research and Ethics Committee linked to the Giresun University (code KAEK-121-06, March 18, 2021).I N FO R M E D CO N S E NT S TATE M E NT Informed consent was obtained from all subjects involved in the study.O RCI D Sevgi Kulaklı https://orcid.org/0000-0001-7886-1060R E FE R E N C E S Clinical, laboratory and ultrasonographic results according to rosacea subtype and severity.
Note: p < 0.05 is defined statistically significant.The bold values indicate statistical significance.Abbreviations: BMI, body mass index; CIMT, carotid intima media thickness; CVD, cardiovascular diseases; DBP, diastolic blood pressure; FA, fetuin A; FBG, fasting blood glucose; HDL, high density lipoprotein; hsCRP, high sensitivity C reactive protein; LDL, low density lipoprotein; MGP, matrix gla protein; n, number; SBP, systolic blood pressure; TC, total cholesterol; WC, waist circumference.a Mean ± standard deviation; b median (min-max); c Pearson chi square test; d Fisher's exact test; e Mann-Whitney U test; f Independent-Samples t-test; TA B L E 1 (Continued) TA B L E 2 Note: p < 0.05 is defined statistically significant.The bold values indicate statistical significance.Abbreviations: BMI, body mass index; CIMT, carotid intima media thickness; DBP, diastolic blood pressure; FA, fetuin A; HDL, high density lipoprotein; hsCRP, high sensitivity C reactive protein; LDL, low density lipoprotein; MGP, matrix gla protein; n, number; SBP, systolic blood pressure; TC, total cholesterol; WC, waist circumference.a Mean ± standard deviation; b median (min-max); c Independent-samples t-test; d Mann-Whitney U-test.