Hypoxia, inflammation, and the occurrence of venous reflux and its interaction with aging



by J. Buján, M. A. Ortega,
C. Mesa-Ciller, F. Sainz, and J. Leal, Spain

Julia BUJÁN,1 MD, PhD
Miguel Ángel ORTEGA,1 MD, PhD
Claudia MESA-CILLER,1 MD, PhD
Felipe SAINZ,2 MD
Javier LEAL,1,3 MD
1Department of Medical Specialties
Faculty of Medicine, University of
Alcalá, Networking Research
Centre on Bioengineering
Biomaterials and Nanomedicine
(CIBER-BBN), Madrid, SPAIN
2Director of Angiology and Vascular
Surgery Service, Central University
Hospital of Defense-UAH
Madrid, SPAIN
3Director of Angiology and Vascular
Surgery Service, International
Ruber Hospital, Madrid
SPAIN

Venous reflux is a clinical sign associated with the inability to control the return of venous flow. Chronic venous insufficiency is subject to change over time. Different factors, such as hypoxia and inflammation, can induce changes in the cytoarchitecture of vein walls, the grade of clinical reflux, and its interaction with aging. The presence of the hypoxia markers—HIF-1α and HIF-2α—and inflammatory markers—IL-6, MMP2, MMP9, and CD206— revealed that patient age affected the severity of reflux in young people (less than 50 years old) and that clinical reflux was related to hypoxic and inflammatory activity in older patients (50 years old or more). Our findings also demonstrated that markers of hypoxia and inflammation increased in the vein walls of patients without apparent clinical reflux, which indicates that histopathological changes occur prior to venous reflux. The presence of HIF-2α, an early marker of hypoxia, was noted in the young population when clinical reflux was not evident. The inflammatory markers exhibited a strong relationship with the severity of vein reflux in the older population.

Medicographia. 2016;38:162-168 (see French abstract on page 168)

The failure of vein wall competence in chronic venous insufficiency (CVI) lacks a clear etiology, despite scientific and technological advances. Multiple factors contribute to its development, including vein wall failure or stretching, valve failure, or valvular agenesis, which are key factors that promote venous reflux.1,2 The measurement of venous reflux assesses the degree of venous involvement in the lower limbs. Clear evidence for a correlation of the severity of reflux with vein wall damage is lacking, which impedes the implementation of any therapeutic measure.

We have previously reported that aging is one of the many insults that affect the morphology and function of the venous wall and contribute to venous failure.3 Venous insufficiency—with or without reflux—dilates the vein wall, which initiates compensatory changes in the structure, including initial hypertrophy in areas that eventually fail, and results in a final fibrosclerotic process that is characteristic of varicose vein walls.4 Inflammation and ischemia induce, encourage, and sustain these alterations, leading to remodeling of the cytoarchitecture of the vein wall that manifests as functional incompetence and is evaluated as venous reflux (Figure 1).

The inflammatory process plays an important role in the aging process and vein wall failure (dilation). The distribution of inflammatory cells3 suggests that dysfunction of the microvascular endothelium is the primary effect of aging, and valve alterations are related to venous insufficiency. Aging and venous insufficiency occur in a parallel, overlapping course, but the aging process may be accelerated in CVI because of the coincident secondary remodeling that is induced by the competency failure.5,6

Figure 1. Representation of the processes that occur during venous remodeling.

CVI induces changes in venous flow return, which may increase venous filling, intraluminal pressure, venous stasis, and relative hypoxia, and these changes are associated with increased oxidative metabolites and reactive oxygen species, which are damaging to the venous wall.7 Nitric oxide is a potent molecular messenger for the regulation of vascular tone and mediation of the inflammatory cascade. Nitric oxide increases the functional activation of monocytes and macrophages8 and activates matrix metalloproteinases (MMPs). MMP2 (gelatinase A) and MMP9 (gelatinase B)—which degrade elastin and collagen gelatinases—and inhibitors of tissue inhibitor of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2 inhibitors) are the most studied enzymes in venous disease. MMP2 and MMP9 are produced in vascular and inflammatory cells.8,9 MMPs play an important role in the synthesis and degradation of the extracellular matrix under physiological and pathological conditions, and any alterations in this balance may lead to a degradation of the matrix with degenerative and structural changes in the venous wall. MMPs also alter muscle and endothelial cells in the absence of severe changes in the extracellular matrix.6,10,11

The level of hypoxia caused by venous hypertension triggers molecular pathways that are involved in the cellular response to the lack of oxygen (O2), such as hypoxia-inducible factor (HIF) transcription factors, which are members of the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) family. HIF is formed by α and β subunits. Three isoforms of the β subunit have been identified, HIF-1α, HIF-2α, and HIF- 3α. The function of HIF-3α is the least characterized. HIF-2α and HIF-1α exhibit a 48% homology and have similar biochemical structures. These factors play a similar role in the induction of gene expression during the hypoxic response, and expression depends on tissue type.12-14 Research suggests that the stabilization of HIF-2α requires less severe hypoxia than HIF-1α, but the mechanisms are not known. HIF-2α may be a first-line response to moderate or less severe declines in O2.15 The involvement of interleukin 6 (IL-6) in the regulation of cell metabolism was also noted recently.16,17 A hypoxic environment produces a significant increase in IL-6 expression, which leads to a positive feedback of the inflammatory response.18

CD206 was observed to play a role in the inflammatory response after IL-6 activation. CD206 is a protein receptor for cysteine in various tissues, and it exhibits a relationship with the functional activity of lectin. This activation plays an essential role in cellular homeostasis as a turning point for the resolution of inflammation. This protein is a marker for the activity of alternatively activated (M2) macrophages, which exhibit a reparative role in chronic inflammation.19,20

Our study investigated the relationship between hypoxia or inflammation—which induce changes in the venous wall— and the grade of clinical reflux and its interaction with aging.

Patients and methods

The study was performed using 30 vein samples from patients undergoing lower-extremity saphenectomy. Patients were divided into three study groups according to the diagnosis of the presence or absence of reflux and the severity grade. Patients were further classified by age—younger vs older (ie, under or over 50 years, respectively). Reflux studies were performed using noninvasive Doppler (7.5-10 MHz color Doppler ultrasonography), which allows adequate exploration of superficial and deep venous systems using the appropriate maneuvers. Patients were classified as having no apparent clinical reflux, moderate vein reflux, or severe vein reflux, as follows:
(i) No apparent clinical reflux (NR): Duration of venous reflux (DVR) 2.0 seconds (n=10). Four patients were under 49 years of age (22-48 years) and six were over 50 (55-73 years). Procedures were followed in accordance with institutional guidelines and conformed to the standards set by the latest revision of the Declaration of Helsinki.

Table I. Immunohistochemical detection of hypoxia-inducible factor (HIF)-1α and HIF-
2α in patients with no apparent reflux, those with moderate reflux, and those with
severe reflux. Results show the percentage of patients staining positively, according
to age group.
Abbreviations: <50, under 50 years of age; >50, over 50 years of age; HIF, hypoxia-inducible factor;
MR, moderate vein reflux; NR, no apparent reflux; SR, severe vein reflux.

Figure 2. Immunohistochemical images showing
the detection of HIF-1α in patients with no
apparent reflux, those with moderate reflux, and
those with severe reflux, by age group.
Abbreviations: <50, under 50 years of age; >50, over 50
years of age; HIF, hypoxia-inducible factor; MR, moderate
vein reflux; NR, no apparent reflux; SR, severe vein reflux.

Immunohistochemical and polymerase chain reaction studies
We used the avidin-biotin complex (ABC) method with alkaline phosphatase as a tracer for immunohistochemical detection of antigens of interest. Samples were washed and balanced in phosphate-buffered saline (PBS), and nonspecific binding sites were blocked for 45 minutes at room temperature in a blocking solution (10% fetal bovine serum [FBS], 1% bovine serum albumin [BSA], and 0.05% Tween 20 in PBS). Samples were incubated with the following primary antibodies overnight at 4°C: rabbit monoclonal anti– human-HIF-1α (1:800) (Abcam, Cambridge, UK), rabbit monoclonal anti–human-HIF-2 α(1:2000) (Abcam, Cambridge, UK), mouse monoclonal anti– human-MMP2 (1:25) (Neomarkers, Fremont, California, USA), rabbit monoclonal anti–human-MMP9 (1:100) (Abcam, Cambridge, UK), and mouse monoclonal anti–human-CD206 (1:1000) in blocking solution. Samples were incubated with the appropriate secondary antibodies bound to biotin for 1.5 hours at room temperature: biotinylated anti– rabbit immunoglobulin G (IgG) (1:1000) (Sigma, St. Louis, Missouri, USA) and biotinylated anti–mouse IgG (1:300) (Sigma, St. Louis, Missouri, USA) in PBS. Samples were incubated with avidin-conjugated alkaline phosphatase (ExtrAvidin–Alkaline Phosphatase, Sigma-Aldrich, St. Louis, Missouri, USA) for 1 hour at room temperature at a 1:200 dilution in PBS. A chromogenic substrate was used for color development under a microscope. A Zeiss Axiophot light microscope equipped with an AxioCam HRc (Carl Zeiss, Oberkochen, Germany) digital camera was used for observations.

Complementary DNA (cDNA) was produced using polymerase chain reaction (PCR) in real time (quantitative RT-PCR), and the amount of cDNA for the following genes was quantified in each sample of interest: IL-6, MMP2, and MMP9. The results were normalized using the constitutively expressed gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Specific primers for all genes studied were designed de novo using the online applications Primer-BLAST21 and AutoDimer.22 RNA extraction was performed according to the method of guanidine isothiocyanate-phenol-chloroform extraction of Chomczynski and Sacchi.23

Quantitative PCR (qPCR) was performed in a StepOnePlus System (Applied Biosystems- Life Technologies, Massachusetts, USA) using the relative standard curve method. A volume of 5 μL of each sample was diluted 1:20 in nuclease-free water, mixed with 10 μL of iQ SYBR Green Supermix (Bio-Rad Laboratories, California, USA), 1 μl of forward primer (IL-6, MMP2, or MMP9; sequences available on request), 1 μL of reverse primer (IL-6, MMP2, or MMP9; sequences available on request) and 3 μL ofdeoxyribonuclease (DNase)- and ribonuclease (RNase)- free water in a 96-well plate (MicroAmp, Applied Biosystems- Life Technologies, Massachusetts, USA), for a total reaction volume of 20 μL. We used Microsoft Office Excel 2010 to analyze the obtained data.

Figure 3. Immunohistochemical images showing
the detection of HIF-2a in patient groups
with higher values of positive expression.
Abbreviations: <50, under 50 years of age; >50, over 50
years of age; HIF, hypoxia-inducible factor; MR, moderate
vein reflux; NR, no apparent reflux; SR, severe vein reflux.

Results

Hypoxia markers
♦ HIF-1 α: Immunohistochemical analysis showed positive staining for HIF-1 α in all patients studied, regardless of reflux grade (Table I, Figure 2). However, in the NR and MR groups, HIF-1 α staining intensity was greater in the younger patients (aged 50 years). The inverse was observed in the SR group, with staining intensity greater in older patients.
♦ HIF-2 α: Statistically significant differences between the study groups were observed. Only 20% of patients in the NR group showed positive staining for HIF-2 α, whereas 100% of patients in the MR group and 40% of patients in the SR group showed staining (Table I, Figure 3). We observed less HIF-2 α staining in patients older than 50 years than in younger patients in the NR and MR groups, a pattern that is reversed in patients with severe vein reflux. The age difference between the younger and older groups also became more significant in the SR group.

Inflammatory markers
♦ MMP2: messenger RNA (mRNA) levels for this marker were clearly different across population profiles according to age and grade of vein reflux. The younger population (Figure 4A). MMP2 mRNA levels in the older patients in the NR and SR groups were similar, but were statistically significantly different from levels in older patients in the MR group. Immunohistochemistry revealeda global immunostaining score of 60% (including both age groups together) for MMP2 protein expression in the NR group, 20% in the MR group, and 80% in the SR group (Figure 5, page 166). Using PCR analysis of cDNA, we observed a similar global mRNA expression regardless of reflux severity (Figure 4D).

Figure 4. (A) Messenger
RNA (mRNA)
levels of MMP2, (B)
MMP9, and (C) IL-6,
quantified using quantitative
real-time polymerase
chain reaction
(qRT-PCR) in patients
with no apparent reflux,
those with moderate
reflux, and those
with severe reflux, according
to age group.
(D) Comparisons of
different mRNA levels
of inflammatory markers
for the three general
groups of patients
quantified using qRTPCR.
Abbreviations: <50, under
50 years of age; >50, over
50 years of age; IL-6, interleukin
6; MMP, matrix
metalloproteinase; MR,
moderate vein reflux; NR,
no apparent reflux; SR,
severe vein reflux.

♦ MMP9: Protein expression was evident (positive staining) in 60% and 100% of the studied patients, according to reflux severity (Figure 5). Gene expression of this marker depended on age. Lower levels of MMP9 protein and mRNA were observed in the younger patients than in the older patients in the NR and SR groups (Figures 4B and 5). Significant differences were observed in mRNA levels of the SR group between the younger and older patient groups (Figure 4B). Overall, higher values correlated with the degree of reflux (Figure 4D).
♦ IL-6: This marker exhibited a direct relationship with age and the grade of vein reflux. Higher values were observed in the SR group. Very low levels were observed in the NR group in the younger population. The presence of this cytokine was higher in the older patients in the NR and SR groups, with the difference reaching significance in the latter (Figure 4C).

A comparison of the overall mRNA expression profiles revealed a clear tendency of an increase in the three inflammatory markers with the degree of vein reflux in our studied population (Figure 4D).

♦ CD206: Immunohistochemical results of CD206 expression, which is involved in M2 macrophage–like activity and other cell types, revealed an interesting pattern with positive staining in smooth muscle cells. A very characteristic patchy pattern of vein wall remodeling was observed. Comparisons by age and grade of reflux revealed significant differences in expression in smooth muscle cells during remodeling, which was more pronounced in the NR group of the young population than in the aged group of the SR population (Figure 6).

Discussion

In this study, patients were classified according to degree of reflux—no apparent clinical reflux, moderate reflux, and severe reflux— which we measured using Doppler ultrasound. Greater reflux severity may correspond to the increasing age of the vein wall. We investigated the role of this blood flow alteration in inducing slowing of venous return and stasis, which promote the development of hypoxia. The evaluation of two markers of the initial state of acute hypoxia—transcription factors HIF-1α and HIF-2α—improved assessment of damage status. We observed the presence of HIF-1α in all patients studied, which reveals a significant state of hypoxia in the venous walls. This hypoxia appeared in the young population— vein stasis triggered a greater degree of hypoxia in young people than older people—and was more marked in the initial stages of venous insufficiency where reflux was not detectable or was moderate. However, hypoxia clearly increased with severe reflux in the patients aged over 50 years.

Figure 5. (Upper panels) Immunohistochemical images showing the detection of
MMP2 and MMP9 in patient groups with a more marked positive expression. (Lower
panel) Results show the percentage of patients staining positively for MMP2 and
MMP9, and according to age group.
Abbreviations: <50, under 50 years of age; >50, over 50 years of age; MMP, matrix metalloproteinase;
MR, moderate vein reflux; NR, no apparent reflux; SR, severe vein reflux.

Figure 6. Immunohistochemical images showing the detection of CD206 in patient
groups with a more marked positive expression. Staining shows the presence of
activated cells expressing these molecules.
Abbreviations: <50, under 50 years of age; >50, over 50 years of age; MMP, matrix metalloproteinase;
MR, moderate vein reflux; NR, no apparent reflux; SR, severe vein reflux.

Our results are in general agreement with authors who observed a significant increase in the expression of the HIF pathway in patients with varicose pathology. The dysregulation of this pathway results in increased angiogenic factors,24-26 and a significant increase in HIF-1α in the muscle layers of diseased vessels has been described. This expression was related to the increased presence of BCL2 (B-cell/CLL lymphoma 2 protein) on the endothelium of the vessel, which leads to an inhibition of apoptosis and increased dilation of the vein wall.

We estimated overall expression of HIF-2α to be lower than HIF-1α, on the basis of immunohistochemical staining observed. We found that in the young patients, HIF-2α expression is lower in those with a severe degree of reflux than in those without severe pathology. Our results are generally consistent with Lim et al,<sup27,28 who found that in varicose veins, HIF-1α and HIF-2α expression was elevated and inversely proportional to the physiological process of hypoxia.

The presence of HIF-1α and HIF-2α overexpression may be a marker of a hypoxic environment at the beginning of the pathological process in young patients. HIF-1α and HIF-2α reach peak expression during moderate reflux and have lower expression when the hypoxic environment becomes severe. HIF factors are short-lived, and ubiquitination inhibits HIF expression and prevents translocation to the nucleus.29 Notably, our study demonstrated that MR is a turning point for adaptation to hypoxia.

The process of cell division is dependent on the availability of O2, but an inhibition of cell proliferation is not always observed in hypoxic situations. Therefore, various O2-independent mechanisms may exist. In this context, HIF-1α, in contrast to HIF-2α, plays a role as a cell attenuator of autonomous proliferation. The amount of O2 is sufficient to produce cellular proliferation in situations of moderate hypoxia; HIF-2α is easily activated because it does not depend on the involvement of many more molecules in this process. An increased expression of HIF-1α is observed in severe hypoxia, which slows cell proliferation.30-32 These changes are compatible with the hypertrophy-atrophy sequences that occur during the remodeling process of the insufficient venous wall.

IL-6 significantly correlated with increasing age in the NR and SR groups. These results concur with previous studies that related this increase with the development of chronic inflammation of the vascular wall.33,34 Notably, expression of IL-6 mRNA in the MR group did not differ between age groups in our study.

One of the important roles of IL-6 involves immunomodulation via the macrophage activation pathway and tumor necrosis factor α (TNF-α).35 Deng et al36 suggested an immunomodulatory role of IL-6 on CD206 in this context. The CD206 activity, characteristic of macrophages, was inversely related to the functional capacity of IL-6, perhaps due to differences in the timing of each protein’s period of activity. That we found CD206 expressed in the smooth muscle cells of the vein wall is not surprising, based on the characteristics of this dimeric cysteine-lectin receptor, which may induce differentiation of mesenchymal cells into smooth muscle cells. The patchy expression pattern of this protein is consistent with observations for previously studied proteins,37 such as elastin and fibrillin- 1. Cells expressing fibrillin-1 and tropoelastin mRNA have been shown to exhibit a patchy disorganized pattern, particularly in the proximal varicose segments of patients under 50 years of age. In that study, enhanced elastase activity was reported in control and varicose samples from older subjects. Varicose vein samples showed greater expression of latent- TGF-β–binding protein 2 (LTBP-2) and TGF-β expression.

The increased MMP2 protein expression in young patients with venous insufficiency corroborated previous work by our group on varicose veins and age.5 MMP9 expression is expected to increase with age and disease, and our results agree with previous studies.9,10 These results support an acute remodeling of the cytoarchitecture of vein walls in young people, which is partially regulated via late MMP2- and MMP9-induced remodeling.

These processes are part of the characteristic events that occur during the final wall remodeling after failure. Our results suggest that the initial moments of vein wall failure may occur in patients even when no reflux is evident and that the events of inflammation-hypoxia-remodeling are triggered in young people and then continue to evolve into a more severe condition over time.

Therefore, factors involved in the process of venous insufficiency are present early, from the first moments of the disease in young patients without clinical symptoms of vein reflux. A treatment plan that is similar to that used in older patients should be implemented in young patients in order to prevent the symptomatology that arises with severe reflux, as immunohistochemical and gene-expression findings reveal a similar pattern in physiological responsiveness during the process of inflammation-hypoxia remodeling. Older patients exhibited a high level of expression of inflammation markers in this study, regardless of the degree of reflux.

Our findings indicate a clear relationship between aging and an inflammation-hypoxia-remodeling process, a process that is irreversible if therapeutic action is not taken.

Acknowledgments. This work was supported by grants from the National Institute of Health Carlos III (FIS-PI13/01513).

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Keywords: hypoxia; inflammation; remodeling; venous insufficiency