The Basic Scientists Are Leading the Way in Understanding, Prevention, and Treatment of Venous and Lymphatic Diseases

Abstract

Purpose Basic science contributes to knowledge and advances the field. This is not an exception in Phlebology. This manuscript highlights the latest contributions to the basic science on varicose veins.

Methods A brief non-systematic review of the literature in the area of basic science and varicose veins was performed using PubMed. We found critical articles that are discussed in this work. In addition, we discussed all the scientific method steps, the importance of each of them, and its application to the field of Phlebology. The basic science and varicose veinsʼ articles were discussed as basic science findings on vein wall, blood flow disturbances, and vein valve changes.

Results Altered vein wall in varicose veins results from the remodeling phenomenon, led by modifications at the vein wall cellular level and in the intercellular space. Smooth Muscle Cells are involved in this process, and a shift between contractile to secretory phenotype has been described. MMPs are actively engaged in the remodeling stage, contributing to the final modification of the vein wall observed in varicose veins. Finally, the blood flow characteristics and the vein valve functionality demonstrated an integrated system.

Conclusions The Scientific Method is the cornerstone of the basic science approach. Varicose veins result from altered blood flow and vein wall remodeling phenomenon.

Keywords

Basic Science - Phlebology - Multidisciplinary Team - Research - Varicose Veins

Introduction

“The current worldwide basic science research efforts on venous disorders are achieving a better understanding, reflected by the recent advances in the phlebology field such as gene mapping, ulcer wound healing, and the genesis of varicose veins. However, we need to acknowledge that there seems to be a gap between our research efforts and how well we translate the current knowledge into the clinical setting to prevent or treat varicose veins” [1] .

The role of basic science, in all medicine areas, was, is, and will be critical. Laboratory studies have been essential in understanding mechanisms that explain the normal function of cells, tissues, and organs and their dysfunction leading to disease status. For instance, basic scientists uncovered the coagulation cascade, which allowed them to discover ways to treat coagulation disorders. Understanding “how it works” in normal conditions also helps understand why a disease occurs. For example, factor V Leiden is a thrombophilia with an increased risk of developing deep vein thrombosis. A group of basic scientists planted the knowledge seed, and other basic scientists grew on the disease knowledge to accomplish such scientific connection on that patient population. All basic science efforts resulted in a current better understanding of our phlebo-lymphology arena, our field.

Basic science and the scientific method

Basic science generates and provides information that is intended to understand the mechanisms involved when natural processes are altered. In other words, basic science explains the “what, why, and how” The investigators dedicated to basic science follow the scientific method ([Fig. 1]). The first step is the observation, which leads to the research questions and literature search to formulate the hypothesis; thus, the basic science projects are, in their vast majority, hypothesis-driven. Once the hypotheses are built, the next step is to formulate a methodology that best test the hypotheses. This step involves designing experiments and may include conducting pilot studies. This phase is considered by the author one of the most complex and important phases of the scientific method. It takes a significant amount of time and effort. Failing a correct design of experiments could potentially drive to inadequate conclusions. Once the experimental design is finalized, the experimentation is initiated in the laboratory, and the results will determine if the hypothesis was confirmed or rejected. This is all about confirming or rejecting a hypothesis. There are no good or bad results, just results.

Fig. 1 The Scientific Method - How we do it. [Fig. 1] illustrates the scientific method with additional steps that basic scientists frequently practice and never mention. Literature search and pilot studies are critical steps during the scientific method process, particularly to define an accurate experimental design.

Application of the scientific method

The observations of the gaps in the knowledge are confronted on a daily bases by researchers, clinicians, and clinical trialists. These observations initiate the scientific method that will improve understanding of a disease process and may directly impact diagnosis and treatments for patients. As an example of the applicability of the scientific method in our specialty, the Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER trial) published in 2009 can be mentioned [2]. The JUPITER trial included more than 17,000 patients with elevated C reactive protein and normal lipids levels. This study was prospective, interventional, randomizing two groups. One group received 20 mg/day of statins (Rosuvastatin) and a control group (placebo), and five years follow up with a mean of 1.9 years. The results demonstrated that over 17,000 patients, 94 developed DVT or PE, and the distribution was 34 in the statin group and 60 in the placebo group (hazard ratio with a statin, 0.57; 95% confidence interval, 0.37 to 0.86; P=0.007) [2]. The authors concluded that statins prevent DVT, but the authors stated at the end of the paragraph before the last: “Overall, validation of our results and further elucidation of the potential mechanisms will be important to confirm our findings” [2]. Basic scientists took this gap (observation) into the lab and developed a basic science project. The first manuscript that brought to light the mechanistic component missing (and requested) by the JUPITER Trial authors was published in 2013 [3]. The authors demonstrated statinsʼ anti-inflammatory and profibrinolytic properties in in-vivo experimentation [3]. Moreover, several manuscripts were published more recently, confirming the 2013 publication results and improving our knowledge of venous thrombosis [3]. Today the non-lipid lowering effects mechanisms of statins and their potential in areas of application that exceed the lipid world are well known, as we discussed later in this manuscript.

Varicose veins, evidence from the basic science

Building a complete understanding of any disease requires a coordinated effort between basic scientists and clinicians of multiple disciplines. “The relative importance and the timing of alterations in vein-wall biology vs. altered flow dynamics. Indeed, altered blood flow can change the demand on the vein wall and initiate biological changes” [4]. On the other hand, changes in vein wall characteristics can promote changes in the lumen flow [4].

Tortuous and non-tortuous vein segments are observed in patients suffering varicose vein problems. Today, we understand that hemodynamic changes in the blood flow and vein wall changes are responsible for the tortuosity and valve incompetency. The question of what is first A) the hemodynamic changes in blood flow within the veins leading to adaptive wall modifications or B) vein wall modifications leading to hemodynamic changes remains unanswered. In this manuscript, we do not intend to solve the mystery. We here instead present the current evidence.

The vein wall

Investigators from basic science demonstrated that those morphological changes on the vein wall were associated with the phenomenon known as tissue remodeling. Tissue remodeling involves a series of biological mechanisms highly regulated. The vein wall is populated by extracellular matrix (ECM) and cells, like any other tissue, and there are changes in both during tissue remodeling.

Extracellular matrix of the vein wall.

Fibrillin-1 is a crucial component of the ECM, with elastics properties. Investigators found increased content levels of Fibrillin-1 in varicose veins [5]. Interestingly, the investigators also explored the skin of those patients and found that Fibrillin-1 was also elevated compared to controls. This type of information supports the influence of genetics on the predisposition to develop varicose veins [5]. In addition, other investigators working on Fibrillin-1 determined that the patientʼs age may affect the remodeling of varicose veins due to the same effect [6].

Collagen is an essential component of the veins. This protein confers physical properties of support resistance and compliance. There are two collagen types as part of vein walls, type I and type III. Type I is more abundant in normal veins. Importantly, investigators demonstrated a shift in those collagen types in varicose veins establishing that collagen type III is the most abundant in the varicose vein walls [7] [8]. The investigators believe that vein walls type I reach are more elastic and contributes to the normal wall activity. On the contrary, abundant type III collagen contributes to vein deformation and less elasticity, both properties that result from adapting to the disease status.

Interestingly, the collagen fibers have a natural disposition to the tridimensional structure of the vein walls that is altered due to tissue remodeling. Despite that, this study was conducted to study bridging veins (BVs), which drain the blood from the cerebral cortex into the Dural sinuses, demonstrating the importance of collagen fiber disposition on those veins after tissue remodeling head impact [9]. Thus, there is a gap in collagen fibersʼ spatial configuration changes in varicose veins and a potential link to risk factors. In summary, the extracellular matrix changes substantially from normal vein to varicose vein status. Those changes indicate active remodeling of the tissue during the disease progression.

Matrix metalloproteinase (MMP) is a group of enzymes that break down collagen and elastin in the extracellular matrix, weakening the vein wall. Thus, they are found in the extracellular matrix. The name “Metallo” is derived from their need to work correctly in the presence of Zinc or calcium. MMP-1, -2, -3, -7, -9 and -13 have been studied in varicose veins. Overexpression of MMP-9 was found in varicose veins, with a positive correlation with elevated levels of vascular endothelial growth factor (VEGF) [10]. VEGF is a molecule (a grow factor) produced by several cells (platelets, macrophages, renal mesangial cells, etc.) that orchestrate tissue remodeling. It is well-known for its participation in vessel formation (angiogenesis) and thus wildly studied in the cancer arena.

Cellular component of the vein wall

In Xu et al., the authors compared the phenotype and functionality of smooth muscle cells (SMCs) from incompetent great saphenous veins (GSV) with normal GSVs (harvested for bypass purposes) [11]. Using fluorescence quantitative polymerase chain reaction and immunoblotting, they tested messenger RNA expression and protein content of Bas, Bcl2, caspase 3, matrix metalloproteinases 2 and 9, and tissue inhibitors of metalloproteinase 1 and 2 on cultured SMCs from normal GSVs vs. incompetent GSVʼs. The authors concluded that SMCs derived from incompetent GSVs increased proliferative and synthetic capacity compared with normal GSVs [11]. In addition, they suggested that the presence of phenotypic and functional differences in SMCs derived from incompetent GSVs may be associated with pathogenesis. The authors bring data supporting the role of vein wall events that could explain the genesis of varicose veins in this cohort of patients. This study provides evidence that incompetent GSVs have phenotypic and functional differences in SMCs; whether this was by the cause of the venous insufficiency or flow alterations caused it remains in question. Building on this work, a biophysical examination of varicose vein pathogenesis may promote convergence on a clinical solution. Thus, basic science work may be translated to the patients, the ultimate beneficiary of our scientistsʼ efforts.

Smooth Muscle Cells (SMC) are found in vein walls in small quantities compared to arteries, but their role is critical to the vein wall and its environment. The SMC plasticity to switch from contractile to secretory phenotype was recently identified. Biologically speaking, phenotype switch means that the cell can be present as a contractile phenotype, providing elastic properties to the vein wall. Also, those cells can be transformed into cells that secret collagen as an adaptation mechanism to the new vein conditions. The importance of switching phenotypes is not only losing the SMC population but also the increase of type III collagen synthesis, as mentioned before. Changing phenotypes and collagen synthesis are part of the same process during the development of varicose veins. Apoptosis is another mechanism of losing the contractile SMC. Together, decreasing the SMC population in the vein wall is today considered a key event in the varicose vein development. Changing phenotype or decreasing cell population are also known as tissue remodeling. Finally, the SMCs can react to hypoxia secreting VEGF.

Guo Z. and collaborators identify that elevated c-fos expression is correlated with phenotypic switching of human vascular smooth muscle cells varicose veins [12]. C-fos is a gene involved in the regulation of cell switching. Varicose vein showed increased mRNA and protein expression of c-fos [12]. Also, Xiao Y. and collaborators demonstrated that SMC switched phenotypes in an in-vitro study [13]. Thus, the authors provided evidence that the alteration of biological behaviors remains present in SMC when they cultured the SMCs derived from varicose veins contributing to the development of varicose veins [13].

The cell switch is not the only mechanism that reduces the number of SMC in the vein wall. Accelerated apoptosis also contributes to decreasing the SMC population. Investigators found that the reduction of the SMC population corresponds to an increase of p21 expression in proximal saphenous vein segments suggesting that the cell cycle disturbances may lead to the “weaknesses” of the proximal GSV wall [14]. The p21 is an inhibitor of a cyclin-dependent kinase, an enzyme that regulates critical metabolic events, including apoptosis.

Finally, Ortega M. and collaborators demonstrated that SMC adapts to hypoxia, secreting VEGF, a core molecule in tissue remodeling [15]. They studied hypoxia markers, including VEGF, starting with the hypothesis that venous insufficiency produces vein dilatation leading to compensatory mechanisms secondary to local hypoxia on the vein wall. SMCs from normal veins produce an elevated secretion of VEGF when exposed to hypoxia. Interestingly, SMCs obtained from varicose veins secrete a large amount of VEGF in normoxia. Understanding the metabolism of venous wall cells under normoxia and hypoxia conditions will help identify possible therapeutic targets, such as VEGF.

Together, tissue remodeling at the cellular and extracellular levels are adaptive events from the environment in normal veins to the one in the varicose condition. All the results presented here from the basic sciences contribute to understanding the biology behind varicose veins and could favor the development of new therapeutic strategies. For instance, Eschrich J et al. from Heidelberg University studied the implicates of statins in preventing the development of varicose veins. The authorʼs results demonstrated that statins inhibit the development of varicose veins by interfering with wall stress-mediated activator protein 1 activity in venous smooth muscle cells. This is a clear example of how the previous knowledge served this group to move the field forward. Being statins an approved medication for other conditions, this work put the basic science very close to patients on preventing recurrent varicose veins [16]. In other words, it is a very translational study, and only clinical research to ensure statin effectiveness in a large population is needed.

Vein Blood Flow

Vein valves and vein flow

Normal circulatory conditions in the veins system (the pulmonary circulation is an exception in adults) are assumed to move low oxygenated blood from the periphery to the right heart. Mechanisms that allow such antigravitational flow are the negative pressure of the thoracic cavity, the low or cero pressure on the right atrium, and the muscle pump on the calf (negative, cero positive). Even though recent questioning of those physical properties of the biological nature of the human body, they exist, and we believe that they contribute to the antigravitational vein blood flow.

In this regard, the presence of vein valves, in good function, helps achieve the ascending antigravitational direction. The absence of vein valves or dysfunction of an existing valve promotes blood to pool in the veins and even causes reflux. Now, valves have pairing arrangements with specific particular orientations. They are paired in an orthogonal configuration. Tien WH et al. characterized the flow interaction of paired valves under controlled in vitro bench conditions [17]. The results of this investigation demonstrated that the 90° valve pairing configuration regulates the flow between the valves, and the separation distance affects the hemodynamic efficiency of the two valves by reducing the total reverse flow volume [17].

Valves favor helicoidal flow within the legʼs veins and have a pairing arrangement with a specific particular orientation. Chen HY et al. improved our understanding of blood flow using a computerized simulation technique [18]. This investigation demonstrated a structure-function relation that optimizes flow patterns in normal physiology, which can be compromised in venous valve disease [18].

Vein valves, reflux, and exercise

The reflux of blood flow, circulating in a reverse direction, even though through valved areas, becomes evident during ultrasonography or in venography when we indicate to a patient to perform a Valsalva maneuver. Although minor reflux can be quantified as normal until valves coapt to stop the reverse flow, continuous reflux in the amount of time and volume is a pattern well characterized on varicose veins. It is believed that valve dysfunction is observed in some histologically determined status, such as valve agenesia, valve involution, or valve that does not coapt in a tortuosity. Either of those determines a valve malfunction, which leads to defective blood circulation and degree of stasis. Even though it is true that valve malfunction determines valve reflux, a description of reflux with correct valve function was described [19], but this subject exceeds the purpose of this manuscript.

Finally, Tauraginskii RA et al. investigate the effect of calf pump activity on ultrasound-measured venous reflux parameters. In this multicenter, prospective experimental study, the authors reported that reflux volume (RV) decreased significantly after physical exercise. The proposed hypothesis is that physical activities change the calf muscle pumpʼs physiologic status, affecting ultrasound venous reflux parameters. The authors included patients with primary incompetence of the great saphenous vein (GSV), and parameters such as the diameter of the GSV, cross-sectional area, time-averaged velocity, and reflux duration were measured by duplex ultrasound examination before and after exercise of 30 lifts to tiptoe. The authors speculated that the decreasing reflux volume was due to exercise-induced hyperemia. Starting from the premise that venous reflux in the great saphenous vein is a retrograde flow returning to the calf venous reservoir through varicose tributaries and perforators of re-entry, the authors postulated that the peripheral pump (calf muscle exercise) promotes hyperemia, which full fill the vein reservoir decreasing the chance to be simultaneously served by the GSV reflux. Thus, the excessive induced arterial hyperemia impacts the calf-deep veins, reducing GSV reflux immediately after the tiptoe exercises. The authors concluded that exercise-induced increased reservoir venous volume reduced venous reflux, highlighting the importance of exercise (calf activation) on venous flow and indirectly decreasing reflux [20].

Multidisciplinary team

The importance of basic science is critical. There is no advance if we donʼt have a basic science discipline filling physiopathology, diagnosis, and treatment gaps. However, the basic science efforts are null if they are not associated with the clinical arena. This association between basic science research and clinical research is named translational research. Thus, we must understand that in todayʼs world, all research communities should embrace the patient problem, and to do that, it is mandatory to work together. A multidisciplinary scientific team is critical to translating the basic science research efforts into the day-to-day battle of clinical practitioners [21]. To set the research priorities, interdisciplinary teams working on consensus and scientific statements on venous and lymphatic diseases are needed [22]. Those types of work can be directional and help the research community set their efforts.

Conclusions and remarks

“Venous and lymphatic diseases, as a subset of cardiovascular diseases, combine organic chemistry, cell and tissue biology, and flow physics events that interact simultaneously in a complex and intimate relationship in both healthy and diseased states. Dissecting the importance of each of these participants in each situation is critical for understanding the mechanisms of disease. However, it is crucial that we carefully consider the biophysics interplay and focus our efforts on understanding the complete clinical scenario, thus ultimately promoting the clinical translation ability of our results” [4].

Interessenkonflikt

Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.

• References

• 1 Diaz JA. Basic-science leading the way for prevention and treatment of varicose veins. J Vasc Surg Venous Lymphat Disord 2021; 9: 252-253
• 2 Glynn RJ, Danielson E, Fonseca FA. et al. A Randomized Trial of Rosuvastatin in the Prevention of Venous Thromboembolism. NEJM 2009; 360: 1851-1861
• 3 Patterson KA, Zhang X, Wrobleski SK. et al. Rosuvastatin reduced deep vein thrombosis in ApoE gene deleted mice with hyperlipidemia through non-lipid lowering effects. Thrombosis Research 2013; 131: 268-276
• 4 Diaz JA. Invited commentary. J Vasc Surg Venous Lymphat Disord 2017; 5: 734
• 5 Robbesom AA, Koenders MMJF, Smits NC. et al. Aberrant fibrillin-1 expression in early emphysematous human lung: a proposed predisposition for emphysema. Modern Pathology 2007; 21: 297-307
• 6 Bastos AN, Alves MMR, Monte-Alto-Costa A. et al. α-smooth muscle actin, fibrillin-1, apoptosis and proliferation detection in primary varicose lower limb veins of women. Int Angiol 2011; 30: 262-271
• 7 Sansilvestri-Morel P, Rupin A, Badier-Commander C. et al. Imbalance in the synthesis of collagen type I and collagen type III in smooth muscle cells derived from human varicose veins. J Vasc Res 2001; 38: 560-568
• 8 Haviarova Z, Janega P, Duedik S. et al. Comparison of collagen subtype I and III presence in varicose and non-varicose vein walls. Bratislava Medical Journal 2008; 109: 102-105
• 9 Kapeliotis M, Gavrila Laic RA, Peñas AJ. et al. Collagen fibre orientation in human bridging veins. Biomechanics and Modeling in Mechanobiology 2020; 19: 2455-2489
• 10 Horecka A, Hordyjewska A, Biernacka J. et al. Intense remodeling of extracellular matrix within the varicose vein: the role of gelatinases and vascular endothelial growth factor. Irish Journal of Medical Science 2021; 190: 255-259
• 11 Xu Y, Bei Y, Li Y. et al. Phenotypic and functional transformation in smooth muscle cells derived from varicose veins. J Vasc Surg Venous Lymphat Disord 2017; 5: 723-733
• 12 Guo Z, Luo C, Zhu T. et al. Elevated c-fos expression is correlated with phenotypic switching of human vascular smooth muscle cells derived from lower limb venous varicosities. J Vasc Surg Venous Lymphat Disord 2021; 9: 242-251
• 13 Xiao Y, Huang Z, Yin H. et al. In vitro differences between smooth muscle cells derived from varicose veins and normal veins. Journal of Vascular Surgery 2009; 50: 1149-1154
• 14 Urbanek T, Skop B, Wiaderkiewicz R. et al. Smooth muscle cell apoptosis in primary varicose veins. European Journal of Vascular and Endovascular Surgery 2004; 28: 600-611
• 15 Ortega MA, Romero B, Asúnsolo Á. et al. Behavior of smooth muscle cells under hypoxic conditions: Possible implications on the varicose vein endothelium. BioMed Research International 2018; 2018: 7156150
• 16 Eschrich J, Meyer R, Kuk H. et al. Varicose Remodeling of Veins Is Suppressed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors. J Am Heart Assoc 2016; 5: e002405
• 17 Tien WH, Chen HY, Berwick ZC. et al. Hemodynamic coupling of a pair of venous valves. J Vasc Surg Venous Lymphat Disord 2014; 2: 303-314
• 18 Chen HY, Diaz JA, Lurie F. et al. Hemodynamics of venous valve pairing and implications on helical flow. J Vasc Surg Venous Lymphat Disord 2018; 6: 517-522
• 19 Lurie F. Anatomical Extent of Venous Reflux. Cardiology and Therapy 2020; 9: 215
• 20 Tauraginskii RA, Simakov S, Borsuk D. et al. The immediate effect of physical activity on ultrasound-derived venous reflux parameters. J Vasc Surg Venous Lymphat Disord 2020; 8: 640-645
• 21 Diaz JA. Vascular surgery and the multidisciplinary vascular research team. Journal of Vascular Surgery: Venous and Lymphatic Disorders 2020; 8: 470-471
• 22 Cushman M, Barnes GD, Creager MA. et al. Venous thromboembolism research priorities: A scientific statement from the american heart association and the international society on thrombosis and haemostasis. Circulation 2020; 142: E85-94

Publication History

Article published online:
10 August 2022