Tissue remodelling and increased DNA damage in patients with incompetent valves in chronic venous insufficiency

Abstract Chronic venous insufficiency (CVI), in which blood return to the heart is impaired, is a prevalent condition worldwide. Valve incompetence is a complication of CVI that results in blood reflux, thereby aggravating venous hypertension. While CVI has a complex course and is known to produce alterations in the vein wall, the underlying pathological mechanisms remain unclear. This study examined the presence of DNA damage, pro‐inflammatory cytokines and extracellular matrix remodelling in CVI‐related valve incompetence. One hundred and ten patients with CVI were reviewed and divided into four groups according to age (<50 and ≥50 years) and a clinical diagnosis of venous reflux indicating venous system valve incompetence (R) (n = 81) or no reflux (NR) (n = 29). In vein specimens (greater saphenous vein) from each group, PARP, IL‐17, COL‐I, COL‐III, MMP‐2 and TIMP‐2 expression levels were determined by RT‐qPCR and immunohistochemistry. The younger patients with valve incompetence showed significantly higher PARP, IL‐17, COL‐I, COL‐III, MMP‐2 and reduced TIMP‐2 expression levels and a higher COL‐I/III ratio. Young CVI patients with venous reflux suffer chronic DNA damage, with consequences at both the local tissue and systemic levels, possibly associated with ageing.


| INTRODUC TI ON
Chronic venous insufficiency (CVI), in which blood return to the heart is impaired, has a high prevalence and incidence worldwide. 1,2 There is a higher incidence of CVI in women than men, 3 and CVI has been related to factors such as pregnancy, hormones, lifestyle, family history, metabolic abnormalities and ageing. 4,5 In western Europe, medical care costs for CVI patients have been estimated at 600-900 million Euros, accounting for 2% of total expenditures. 6 To understand CVI, it must be viewed as a cause of venous hypertension whose most characteristic clinical manifestation is the appearance of varicose veins. 3,7 A common complication of CVI is the development of valve dysfunction or incompetence that produces blood reflux, which aggravates venous hypertension and increases hydrostatic pressure. 8 CVI is a complex disease in which the vein wall undergoes changes, although the pathological mechanisms involved in the homeostatic imbalance of CVI remain unclear. Alterations in the extracellular matrix (ECM) have been shown to be essential in this venous disease. 9,10 Collagen fibres play an important role by participating in the homeostasis of vascular cells (endothelial cells, muscle cells and fibroblasts). 11,12 In the vascular system, collagens I (COL-I) and III (COL-III) are found in greater proportions and are modified depending on the pathology. 13 The ECM undergoes permanent remodelling involving the degradation and synthesis of new proteins by proteases such as metalloproteinases (MMPs). 14 The structural and functional diversity of MMPs rival those of the collagen superfamily, and MMP-2 has a prominent role in vascular remodelling. [15][16][17] The proteolytic activity of MMP-2 is regulated at three levels: first-via expression and secretion; second-through pro-enzyme activation and third-through inhibition by tissue inhibitors of MMPs (TIMPs). This regulation serves to maintain a delicate balance. 18,19 Changes in the vein wall lead to an inflammatory process in which pro-inflammatory cytokines trigger cellular responses. 20 -24 Recent studies have revealed the presence of an oxidative stress environment that has a direct effect on signal transduction pathways in patients with CVI, with consequences at both the systemic and vein wall levels. [25][26][27] Moreover, metabolic and chronic disorders can lead to DNA damage, inducing an increase in the activity of repair molecules such as poly(ADP-ribose) polymerase (PARP). 28,29 The aims of this study were to examine the consequences of CVI on the venous wall for a better understanding of the pathophysiological consequences for analysing the (a) possible alterations in PARP expression, (b) the role of pro-inflammatory cytokines such as IL-17 and (c) ECM remodelling in CVI patients with valve incompetence (venous reflux). These factors were also examined in relation to patient age.

| Study population
One hundred and ten patients were divided by age (cut-off, 50

| Diagnosis by imaging
The patients were performed using a 7.5-Mz M Turbo Transducer Echo-Doppler (SonoSite). The lower limb examination was performed in the includes the full spectrum of morphologic and functional abnormalities of the venous system, from telangiectasies to venous ulcers. Patients have been a CEAP classification C1 or higher (Table S1).

| Immunohistological studies
After the necessary time in the fixation solution (10 days), the samples were dehydrated and processed according to standardized protocols. 25 Then, paraffin blocks were made using moulds. Once the paraffin solidified, a rotation microtome HM 350 S (Thermo Fisher Scientific) was used to cut 5μm-thick sections, which were stretched in a hot water bath and collected on glass slides coated with 10% poly-L-lysine to facilitate tissue bonding. Antigen-antibody reactions were detected using the avidin-biotin complex (ABC) method with specific primary (Table 1A) and secondary antibodies (Table 1B)

| Sirius red staining
This technique distinguishes immature collagen (COL-III), which appears yellowish-green, from mature collagen (COL-I), which appears reddish. Staining is observed under a dark-field microscope.
The following protocol was utilized as follows: (a) Sirius red staining for 30 minutes; (b) wash with running water; (c) dehydration in  All the assays were performed in duplicate (Table S2).

| Statistical analysis and interpretation of the results
For statistical analyses, GraphPad Prism ® 5.1 software was used with Mann-Whitney U test. The data are presented as the median with interquartile range (IQR). Significance was set at P < .05 (*) and P < .005 (**). For each patient in the established groups, five sections and 10 fields per section were randomly selected and examined. Patients were characterized as positive when the average marked area in the sample analysed was greater than or equal to 5% of the total area, following the anatomicalpathological protocol of Cristóbal et al 30 In each sample, immunohistochemical staining was scored using the following scale: 0-1, minimum staining (0%-25%); 2, moderate staining (25%-65%); 3, strong staining (65%-100%). This procedure is a minimal modification of the immunoreactive score (ISR score). 30 The preparations were examined under a Zeiss Axiophot light microscope (Carl Zeiss) equipped with an AxioCam HRc digital camera (Carl Zeiss).

| Patients with valve incompetence show elevated PPAR expression in the vein wall
The qRT-PCR showed a significant increase in PARP gene expres-     Figure 2B and Table. S2)].

| Valve incompetence causes an increase in the expression of pro-inflammatory cytokines (IL-17)
IL-17 protein expression was positive in 100% of R patients Interestingly, R patients showed particularly intense IL-17 expression in smooth muscle cells ( Figure 2E,F) TGA AGG TCG  GAG TCA   GTC ATT GAT GGC AAC  AAT ATC CAC T   60°C   PARP  CCA GGA TGA AG AGG  CAG TGA AG   TTC TGA AGG TCG ATC  TCA TAC TCC   58°C   IL-    in the ECM of the three tunica, as well as in the intensity cytoplasm of smooth muscle cells ( Figure 5C-F). Notably, the expression was higher in the R < 50 group than in the other study groups ( Figure 5E).  Figure 6B and Histological analyses showed that TIMP-2 extended through the three tunica of the vein wall in the patients with positive expression.

| Young patients with valve incompetence show elevated MMP-2 expression and reduced TIMP-2 expression
Remarkably, in NR < 50 patients, TIMP-2 expression appeared as accumulations ( Figure 6C, asterisk). In R ≥ 50 patients, high-intensity TIMP-2 expression was present in the three tunics of the vein, characteristic of findings in smooth muscle cells ( Figure 6F,G). Patients in the other groups had mild-intensity TIMP-2 expression, and the majority of the samples were negative ( Figure 6). Immunoreactive score was lower in R < 50 patients (

| D ISCUSS I ON
Over time, it has become accepted that venous pathology, and specifically chronic venous diseases such as varicose veins, develop as a result of increased pressure. 31,32 Current studies suggest that a set of factors act on this disease, causing changes in intrinsic structure and collagen content that play an important role in the aetiology of CVI. 31,33 Previous studies have shown interactions between different vein wall components, cells and especially collagen, demonstrating their indispensable role in the maintenance of venous tone and compression; moreover, ageing is known to produce changes in the vein wall structure. 34 In this sense, our results indicate that valve incompetence increases ECM remodelling, manifested by alterations in COL-I and COL-III levels in the vein wall, and show that this process is  with a study of larger sample size and age that allows a multivariate analysis to be carried out with comorbidities and specific pathophysiological changes, but we provide a detailed histological study of this protein expression.

| CON CLUS IONS
Our findings indicate that patients with valve incompetence in the context of CVI undergo chronic expression of related protein with DNA damage, which has potential consequences not only at the local tissue level but also at the systemic level, possibly in association with ageing.

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

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