Servier – Phlebolymphology

Abstract

This article reviews the literature on microcirculatory disorders underlying the development of chronic venous diseases (CVD) across all the CEAP (Clinical-Etiology- Anatomy-Pathophysiology) clinical classes from C0s to C6 and the fundamentals of their systemic pharmacological correction particularly with micronized purified flavonoid fraction (MPFF). Anatomical and functional changes specifically in the vessels of the microvasculature are the main pathogenetic mechanism for the development of most vein-specific symptoms and determine CVD progression. The altered vessels of the microvasculature are characterized by valvular incompetence and tortuosity that make them similar to glomerular capillaries. The main morphological alterations are a decrease in the functional capillary density; an increase in the dermal papilla diameter, capillary glomerulus, and capillary diameter; as well as an increase in the ratio of abnormal capillaries. These early changes are already observed at clinical class C0s. Atypical microcirculatory vessels lose their ability to maintain venoarteriolar reflex and vasomotor function. Increased vascular wall permeability leads to formation of perivascular extravasates. Inflammation and congestion decrease transcutaneous O₂ pressure and increase CO₂ pressure, which is associated with an increased generation of free radicals and triggers of tissue damage. Evidence from clinical and experimental studies suggests that MPFF can reduce permeability and diameter of microvasculature vessels, modulate leukocyte-endothelial interactions and, therefore, reduce leukocyte activation and vein-specific inflammation by inhibiting the secretion of adhesion molecules and proinflammatory cytokines. In addition, MPFF has free radical scavenging properties and increases venous contractility. These properties substantiate a high efficacy of MPFF and strong recommendation for its use in the recent international CVD guidelines for the treatment of pain, heaviness, feeling of swelling, functional discomfort, cramps, leg redness, skin changes, edema and quality of life, as well as for the healing of leg ulcers in patients with CVD. Given the scarce data on the reversibility of microcirculatory changes in the management of CVD, it is reasonable to consider MPFF in the management of CVD patients.

Introduction

Chronic venous diseases (CVD) refer to a diverse group of morphological or functional abnormalities of the venous system affecting deep, superficial, and/or intradermal veins.¹ Despite the etiological heterogeneity, all CVD forms share a common pathogenesis, in particular, the microcirculatory alterations, which underlie the development of venous symptoms and the progression of skin trophic disorders.² Microvasculature changes have a number of typical morphological and functional features. In general, morphological alterations are similar to those in the saphenous veins and are manifested by the vein tortuosity and formation of venous reflux, which can occur in this vascular territory with or without the concomitant hemodynamic changes in the saphenous veins.³

Studies have revealed the presence of valves in the arteriovenous anastomoses at the level of postcapillary venules, as well as efferent venules, with a typically bicuspid structure, although in rare cases the unicuspid and tricuspid valves have been described.⁴ In the lower limbs, valves are found in the veins with a diameter of greater than 18-20 μm.^1,4 Structurally, the valves are composed of 2 layers of endothelial lining on the basal membrane consisting of collagen fibers.⁴

In a study with retrograde venous filling of amputated lower limbs with a contrast media, microvalves were identified down to the sixth generation of tributaries from the great saphenous vein (GSV).³ They were most prevalent in the third generation of tributaries, constituting the “boundary” microvalves that prevented reflux extension into the microvenous networks in the skin. In addition, the third and higher generation veins without valves were identified, which played the role of collaterals, shunting blood flow to the distal branches of the capillary bed bypassing the competent veins.³

In the lower limbs with signs of severe chronic venous insufficiency (CVI), the reflux of contrast media was extended down to the venules of the capillary network of the skin, which had a dilated tortuous structure and stretched valves.³ The advanced classes of CVI were characterized by retrograde blood flow in both the conducting veins and skin venules.

In a number of patients, reflux in the capillary network in the skin is observed even if GSV is competent.³ Instrumental studies have shown that the occurrence of venous symptoms in patients with clinical class C0s is associated with an isolated retrograde blood flow in the microvasculature vessels, with retention of residual venous volume in them.² In such patients, venous occlusion plethysmography reveals the reduced emptying of the venous reservoir and reduced venous refilling time, compared with healthy individuals.²

As CVD progresses from low to high grade, the number of functional capillaries in the skin decreases.^5,6 The remaining capillaries acquire a tortuous shape with a large number of loops, which makes them similar to the renal glomeruli.^5,6 The transition from C0 to C5 class was shown to be associated with the reduction in the number of capillaries amenable for the assessment from 8 (5-10) to 4 (2-5), whereas the number of convoluted loops in capillary glomeruli increases from 1 to 8 (5-10).⁵ In the center of white atrophy spots, no capillaries are visible (avascular fields).⁶

Orthogonal polarization spectral (OPS) imaging is a method used to quantify morphological features of glomerulus-like capillaries.⁷ The CVD progression from C1 to C5 class was found to be associated with a reduction in the functional capillary density (FCD), ie, the density of capillaries with flowing red cells (from 20.9 ± 6.1 to 12.1 ± 8.1 cap/mm²) and an increase in the diameter of dermal papilla (from 111.4 ± 13.5 to 223.9 ± 126.9 μm), diameter of capillary bulk (from 52.8 ± 8.8 to 149.1 ± 56.3 μm), and capillary diameter (from 8.1 ± 0.8 to 11.1 ± 2.9 μm), as well as with changes in capillary morphology (% of abnormal capillaries within the field of view; from 3.6 ± 5.5% to 75.2 ± 37%).⁷

Comparison of atypical capillaries revealed significant differences in the above parameters between patients with clinical class C1 and healthy individuals.⁷ Moreover, the alterations were also observed in patients with clinical class C0s. Compared with healthy individuals, the latter group had a significantly lower FCD and a significantly greater diameter of dermal papilla, which can be considered the first morphological response to the development of venous hypertension.¹ In addition, there was a trend toward an increase in the diameters of the capillary bulk and of capillaries; however, this finding did not reach statistical significance versus healthy controls.¹

Structural changes in the microvasculature vessels are associated with the loss of a number of their functions, including the ability to produce the venoarteriolar reflex (VAR), which explains an increase in intradermal blood flow in the orthostatic and sitting positions.² VAR is a local protective axonal reflex with arteriolar vasoconstriction in response to a change in the body position, which provides a decrease in cutaneous blood flow by 40% to 50% or more and prevents the development of hypertension and edema at the microcirculatory level.^2,8 The laser Doppler flowmetry shows a reduction in the difference in microcirculatory blood filling between the supine and orthostatic positions by up to 30% already in clinical class C0s.²

Dysfunction of microcirculation vessels results in a reduction in their vasomotor activity, as evidenced by laser Doppler flowmetry data. In patients with CVD, the microcirculatory index, which depends on the number of red blood cells (RBCs) reflecting the laser beam, is significantly increased due to venous stasis.^9,10 The skin flux (the concentration of moving blood cells multiplied by the magnitude of the median velocity) also decreased and became smaller with CVD progression and at higher clinical class levels.10 Starting from C4 class, these variations in some patients can be smoothed to a straight line.¹⁰

Atypical capillaries show increased wall permeability, as determined by microscopy and verified by intravenous administration of a fluorescent dye.⁶ With light microscopy, a “cobblestone pavement” pattern is clearly visualized around the altered capillaries, which is explained by various diameter extravasates of capillary bed contents, consisting of fibrin/fibrinogen, other proteins, and polysaccharides.^9,11,12 Hyperpigmentation can be observed along the edges of such “halo” due to accumulation of hemosiderin, a product of hemoglobin degradation.⁹ The injected fluorescent dye, spreading beyond the capillary wall, creates a high-intensity glow in the pericapillary space, which is most pronounced in patients with CVD of clinical class C3 or higher.⁹ At the same time, a high concentration of the dye is achieved much faster than in healthy individuals. Differences in the diameter of the glow zone were also observed (138 ± 13 μm and 81 ± 15 μm, respectively).

In severe CVI, the filling of capillaries with a fluorescent dye in the observation area is slowed down, which is explained by the inhomogeneity of the perfusion of microvasculature.⁹ In some areas, the distribution of contrast agent is halted due to the occurrence of RBC sludges, which may be explained by microthromboses.^9,12 The latter, in turn, can result in micronecroses in the perivascular space.¹²

Pericapillary edema, as a consequence of high permeability of the vascular wall, creates conditions for an increase in the intraneural pressure in adjacent nerves, which, in turn, activates their alpha fibers and leads to the occurrence of pain and a feeling of heaviness in the lower limbs.¹³

Blood congestion in the abnormal vessels of the microvasculature is accompanied by a decrease in the transcutaneous oxygen pressure (TcPO2), an increase in the transcutaneous carbon dioxide pressure (TcPCO₂), and high concentrations of free oxygen radicals.⁶ The TcPO₂ is measured using the Clark electrode. The current on the probe exposed to the tissue oxygen is measured and is proportional to the oxygen content in capillaries.⁶ A reduction in the TcPO2 from 56.8 ± 9.9 to 47.7 ± 14.5 mm Hg was revealed in patients with CVD, compared with healthy individuals. The reduction was even greater (down to 22.5 ± 7.0 mm Hg) and achieved statistical significance in patients with trophic changes (hyperpigmentation, lipodermatosclerosis, healed trophic ulcer). In the late stages of CVI with the development of lipodermatosclerosis, there is an increase in type IV collagen synthesis, which results in the vessel wall thickening and an increase in its permeability.¹⁴ The concomitant fibrosis in the pericapillary space creates a barrier preventing transmembrane diffusion.¹⁵ Disturbance of microcirculatory tissue perfusion is associated with an increase in TcPCO₂.^16,17 One of the effects of CO2 is vasodilation of capillaries, which contributes to the further progression of stasis and results in an even more significant increase in CO2.¹⁶ The reduction in stasis during topical treatment with a venoactive combination agent (escin + heparin + essential phospholipids) results in an improvement in tissue perfusion, which is manifested by an increase in TcPO₂ and a decrease in TcPCO₂.^16,17 The severity of venous symptoms correlates with these parameters and decreases with the improvement in tissue perfusion.¹⁸

Venous stasis and hypoxia are associated with an increase in expression of plasma free radicals (PFRs) in capillary blood, which are considered one of the triggers of tissue damage and which slow down tissue repair.¹⁹ The major source of PFRs are leukocytes, which are active participants in inflammation of the vascular wall in microvasculature in patients with CVD.²⁰ Spectroscopy of blood obtained from the skin puncture site in the area of interest showed that concentration of oxidation products in patients with high ambulatory pressure and low venous refilling time is significantly higher than in healthy individuals.^19,21 The treatment targeted at improving microcirculation is associated with changes in PFR levels.²¹ Studies have demonstrated a reduction in the PFR level in patients who received compression therapy (stockings with compression level of 20 mm Hg) in combination with a venoactive drug (VAD), or topical therapy with venoactive combination agent (escin + heparin + essential phospholipids).^19,21 In patients with CVD, a significant difference from baseline was reported after 2 and 4 weeks of treatment.^19,21 The reduction in PFRs was associated with a decrease in the severity of venous symptoms, such as edema, pain, feeling of swelling, and restless legs.²¹

Pharmacological effects in microcirculatory disorders

Experimental animal models of venous hypertension have been developed to study pathophysiological mechanisms underlying CVI, as well as to investigate opportunities for pharmacological correction. The animal model most closely representing the pathogenesis of venous hypertension is a rodent (hamster) model based on external iliac vein ligation, which results in substantial changes in saphenous veins without inducing systemic fluctuations in venous pressure.²⁰ All changes were significant compared with those in hamsters after sham surgery without vein ligation. Chronic venous hypertension reaches its maximum severity in 6 to 10 weeks and is manifested by an increased pressure in the saphenous veins, decreased number of functional capillaries (with preserved blood flow), as well as signs of intensive rolling of leukocytes, their adhesion to the walls of capillaries, and dilation of venules while maintaining the diameter of arterioles. In this rodent model, oral administration of MPFF or diosmin alone was found to be effective in reducing these changes.²⁰ During the pharmacotherapy, a decrease in the rolling and adhesion of leukocytes and an increase in the number of functional capillaries were observed. The beneficial effects of MPFF were significantly greater than with diosmin alone, and only MPFF provided a decrease in the diameter of venules in venous hypertension.

The MPFF effect on the microvascular function can also be assessed using the models of angiopathy in other vascular territories. Stimulation of the vasculature of hamster cheek pouch by the application of bradykinin or histamine for 5 minutes causes abundant diffusion of the fluorescent dye through the vascular wall.²² The effects of systemic therapy with MPFF for 10 days compete with the vascular effect of topical agents, contributing to a decrease in the permeability of postcapillary venules.²² The same effect of MPFF is also observed in the experiment with reperfusion of the microvascular bed of hamster cheek pouch after 30-minute ischemia induced by clamping of the main feeding artery.^22,23 A reduction in the venular permeability is associated with a decrease in leukocyte adhesion to the venular endothelium.^22,23

In the model of ischemia-reperfusion in the hamster skin flap, the MPFF treatment was associated with a weak adhesion of leukocytes to the endothelium of venules, compared with controls without the MPFF treatment.²⁴ This effect of the drug prevails over the hemodynamic effect, as evidenced by the absence of changes in the parameters of blood flow velocity in the venules of the skin flap after reperfusion.²⁴

In mesenteric venous hypertension, the effects of MPFF are similar to those in the CVD model.²⁵ Thus, 1-week treatment with MPFF in rats resulted in a faster return of the diameter of venules to normal values during reperfusion of the mesenteric territory, without changing the blood flow velocity parameters. Besides the inhibition of local leukocyte-endothelial adhesion in the experiment, the anti-inflammatory action of the drug is manifested by a systemic reduction in the activity of circulating leukocytes, with suppression of CD62L gene expression, a decrease in pseudopodia formation, and negative results for a nitro blue tetrazolium (NBT) test.

MPFF also modulates leukocyte-endothelial adhesion in postcapillary venules of skeletal muscles after ischemia, accompanied by an increase in levels of adhesion molecules (P-selectin and intercellular adhesion molecule 1 [ICAM-1]),²⁶ and results in a decreased rolling and adhesion of leukocytes in the damaged muscle.

In an experiment with injection of a sclerosing agent in the dorsal vein of a rabbit ear, the effect of MPFF on microcirculation was manifested by a decrease in the diameter of venules, an increase in the number of functional capillaries, and a decrease in their permeability.²⁷ This experiment has also demonstrated the characteristic effect of reducing leukocyte rolling and adhesion. The MPFF effect on the capillary bed is manifested by a reduction in vascular permeability, an improvement in vascular resistance, a decrease in blood stasis, and an increase in blood flow and RBC flow rates.^28,29

In clinical practice, MPFF administration in patients with clinical class C1 of CVD undergoing sclerotherapy alleviates the local inflammatory response to the procedure.³⁰ This is confirmed by a significant decrease in the local concentration of inflammatory markers (C-reactive protein, interleukin [IL]-1, tumor necrosis factor [TNF], vascular endothelial growth factor [VEGF], and histamine), compared with the control group without MPFF.³⁰ The VEIN ACT PROLONGED-C1 observational program (Administration of Micronized Purified Flavonoid Fraction During Sclerotherapy of Reticular Veins and Telangiectasias) has shown that in patients undergoing sclerotherapy, MPFF treatment is associated with an improvement in the patient’s quality of life (QOL), and a significant decrease in sensation of leg heaviness, pain, swelling, and itching.³¹ Similar clinical results were obtained in the SYNERGY survey, which included patients with CVD of clinical classes C1-C3.³² In patients undergoing sclerotherapy, MPFF administration allowed an achievement of treatment satisfaction in 81% of patients, not only in terms of a cosmetic effect of the procedure, but also in reduction in the severity of venous symptoms.

The treatment efficacy of MPFF has been demonstrated in a large number of studies, which provided grade A evidence for the use of MPFF in monotherapy for CVD.³³ MPFF treatment is associated with a reduction in the severity of leg pain and heaviness, feeling of swelling, nocturnal cramps, and edema of the lower limbs, as well as with a QOL improvement both in clinical classes C0s-C1s and in CVI, including those with trophic disorders up to active venous ulcers (C4 and C6).^33-35 The efficacy of MPFF as regards accelerating ulcer healing in the comprehensive treatment of venous ulcers is determined by improvement of microcirculation and reduction in venous inflammation, as demonstrated in a meta-analysis.³⁶

Pathogenetic basis of microcirculatory disorders in CVD and the MPFF action

Blood reflux, stasis, and tissue hypoxia at the microcirculation level determine changes in the activity of the endothelium, with modulation of the expression of adhesion molecules on endothelial cells, including vascular cell adhesion molecule 1 (VCAM-1), ICAM-1, lymphocyte function-associated antigen 1 (LFA-1), and very late antigen 4 (VLA-4).^28,37,38 Changes in endothelial cell phenotype result in an increased adhesion of leukocytes and their activation. Activated leukocytes are a source of enzymes and oxygen free radicals that are released into the environment.³⁹ Unlike saphenous veins, capillaries do not have a typical 3-layer wall structure, and as a result, lytic enzymes of leukocytes destroy subendothelial and pericapillary structures.²⁸ Activated cells secrete various cytokines, including IL-8; regulated on activation, normal T-cell expressed and secreted (RANTES; also known as chemokine [C-C motif] ligand 5); monocyte chemoattractant protein-1 (MCP-1); macrophage inflammatory protein 1beta (MIP-1ß); and VEGF.⁴⁰ A variety of cytokines, which are secreted by activated leukocytes and endothelial cells, contributes to the activation of fibroblasts and further attraction of monocytes and mast cells, which are also a source of enzymes and mediators.^28,39 One of the significant mechanisms of damage to the capillary wall is the destruction of extracellular matrix by matrix metalloproteinases (MMP), which are secreted by activated leukocytes, endothelial cells, and other cells.^41,42 MMPs destroy both collagen and elastin⁴³ and also damage glycocalyx on the endothelial surface, which results in an exposure of adhesion receptors and increased leukocyte endothelial adhesion.⁴¹

Changing properties of the endothelium–due to its activation and cytokine aggression, as well as enzymatic damage to subendothelial structures–result in an increase in capillary permeability, with the possibility of extravasation of not only plasma, but also large molecules.^28,39

MPFF has anti-inflammatory properties, reduces vein-specific inflammation in the vessel wall, and has a protective effect on the surrounding parenchyma.²⁵ MPFF was shown to decrease expression of adhesion molecule CD62L on leukocytes and levels of plasma-soluble markers of endothelial activation– sVCAM-1, sICAM-1–which results in modulating the endothelial-leukocyte interactions without affecting leukocyte function and provoking leukopenia.^44-47 In patients with trophic changes of skin, MPFF decreases the levels of lactoferrin and VEGF; normalizes the levels of prostaglandins E2, F2, and thromboxane B2; and suppresses platelet activation.^28,45,48

Treatment with MPFF suppresses the production of oxygen free radicals by activated polymorphonuclear neutrophils and macrophages, which contributes to a decrease in capillary permeability and a damaging effect on tissues.^49,50

An experiment has shown that in veins of a larger caliber containing a muscle layer, MPFF treatment prolongs the action of norepinephrine and increases the sensitivity of the contractile apparatus of the vascular wall to Ca²⁺, as well as venous contractions.^51-53 The restoration of normal venous tone in large veins can indirectly reduce blood stasis in the microvasculature and, therefore, the severity of clinical manifestations of venous symptoms.⁵⁴

Conclusion

Microcirculatory disorders underlie the development of CVD and its progression across all its clinical classes. Anatomical and functional changes specifically in the vessels of the microvasculature are the main pathogenetic basis for the development of most vein-specific symptoms and determine CVD progression. Treatment aimed at elimination of microcirculatory disorders is associated with an improvement in the QOL of patients and a decrease in the severity of CVD symptoms. Today, the effects of MPFF in the treatment of CVD have been assessed in a large number of clinical and experimental studies, clearly demonstrating its undoubted efficacy. In recent international CVD guidelines, MPFF is strongly recommended for the treatment of pain, heaviness, sensation of swelling, functional discomfort, cramps, leg redness, skin changes, edema, and QOL, as well as for the healing of leg ulcers in patients with CVD.³³ When choosing treatment strategy in patients with any class of CVD, it is necessary to consider the lack of data on the reversibility of microcirculatory disorders on top of any conservative or surgical treatment. The probable persistence of changes in the microvasculature may cause relapses in venous symptoms and indicate the need for regular supportive courses of conservative therapy with VADs, primarily with MPFF.





REFERENCES
1. Senra Barros B, Kakkos S, De Maeseneer M, Nicolaides A. Chronic venous disease: from symptoms to microcirculation. Int Angiol. 2019;38(3):211-218.
2. Nicolaides A, Bouskela E. Pathophysiological mechanisms of chronic venous disease and their role in C0s clinical class. Vasc Invest Ther. 2018;1:103- 109.
3. Vincent JR, Jones GT, Hill GB, van Rij AM. Failure of microvenous valves in small superficial veins is a key to the skin changes of venous insufficiency. J Vasc Surg. 2011;54(6 suppl):62S-69S.e1-e3.
4. Caggiati A, Phillips M, Lametschwandtner A, Allegra C. Valves in small veins and venules. Eur J Vasc Endovasc Surg. 2006;32(4):447-452.
5. Howlader M, Smith P. Microangiopathy in chronic venous insufficiency: quantitative assessment by capillary microscopy. Eur J Vasc Endovasc Surg. 2003;26(3):325-331.
6. Franzeck UK, Haselbach P, Speiser D, Bollinger A. Microangiopathy of cutaneous blood and lymphatic capillaries in chronic venous insufficiency (CVI). Yale J Biol Med. 1993;66(1):37-46.
7. Virgini-Magalhães C, Porto C, Fernandes F, Dorigo D, Bottino D, Bouskela E. Use of microcirculatory parameters to evaluate chronic venous insufficiency. J Vasc Surg. 2006;43(5):1037-1044.
8. Silva H, Ferreira HA, da Silva HP, Monteiro Rodrigues L. The venoarteriolar reflex significantly reduces contralateral perfusion as part of the lower limb circulatory homeostasis in vivo. Front Physiol. 2018;9:1123.
9. Leu AJ, Leu HJ, Franzeck UK, Bollinger A. Microvascular changes in chronic venous insufficiency–a review. Cardiovasc Surg. 1995;3(3):237-245.
10. Azizov GA, Kozlov VI. Chronic venous insufficiency of the lower limbs: microcirculatory features [in Russian]. RUDN J Med. 2003;3:117-120.
11. Fagrell B. Vital microscopy and the pathophysiology of deep venous insufficiency. Int Angiol. 1995;14(3 suppl 1):18-22.
12. Leu HJ. Morphology of chronic venous insufficiency–light and electron microscopic examinations. Vasa. 1991;20(4):330-342.
13. Jawien A, Bouskela E, Allaert FA, Nicolaïdes AN. The place of Ruscus extract, hesperidin methyl chalcone, and vitamin C in the management of chronic venous disease. Int Angiol. 2017;36(1):31- 41.
14. Neumann HA, Van den Broek MJ. Increased collagen IV layer in the basal membrane area of the capillaries in severe chronic venous insufficiency. Vasa. 1991;20(1):26-29.
15. Brakman M, Faber WR, Kerckhaert JA, Kraaijenhagen RJ, Hart HC. Immunofluorescence studies of atrophie blanche with antibodies against fibrinogen, fibrin, plasminogen activator inhibitor, factor VIII-related antigen, and collagen type IV. Vasa. 1992;21(2):143- 148.
16. Incandela L, Belcaro G, Cesarone MR, De Sanctis MT, Griffin M. Microangiopathy and venous ulceration: topical treatment with Essaven gel–a placebocontrolled, randomized study. Angiology. 2001;52(suppl 3):S17-S21.
17. Incandela L, Belcaro G, Nicolaides AN, Geroulakos G, Cesarone MR, De Sanctis MT. Microcirculation after standardized application of Essaven gel on normal skin- -a placebo-controlled, randomized study. Angiology. 2001;52(suppl 3):S5-S10.
18. Ruffini I, Belcaro G, Cesarone M, Dugall M. Efficacy of topical treatment with aescin + essential phospholipids gel in venous insufficiency and hypertension. Angiology. 2004;55(6 suppl):S19-S21.
19. Ricci A, Ruffini I, Cesarone MR, et al. Variations in plasma free radicals with topical aescin + essential phospholipids gel in venous hypertension: new clinical data. Angiology. 2004;55(6 suppl):S11-S14.
20. das Graças C de Souza M, Cyrino FZ, de Carvalho JJ, Blanc-Guillemaud V, Bouskela E. Protective effects of micronized purified flavonoid fraction (MPFF) on a novel experimental model of chronic venous hypertension. Eur J Vasc Endovasc Surg. 2018;55(5):694-702.
21. Cesarone MR, Incandela L, DeSanctis MT, Belcaro G, Dugall M, Acerbi G. Variations in plasma free radicals in patients with venous hypertension with HR (Paroven, Venoruton; 0-(beta-hydroxyethyl)-rutosides): a clinical, prospective, placebo-controlled, randomized trial. J Cardiovasc Pharmacol Ther. 2002;7(suppl 1):S25-S28.
22. Bouskela E, Donyo KA. Effects of oral administration of purified micronized flavonoid fraction on increased microvascular permeability induced by various agents and on ischemia/ reperfusion in the hamster cheek pouch. Angiology. 1997;48(5):391-399.
23. Cyrino FZ, Bottino DA, Lerond L, Bouskela E. Micronization enhances the protective effect of purified flavonoid fraction against postischaemic microvascular injury in the hamster cheek pouch. Clin Exp Pharmacol Physiol. 2004;31(3):159-162.
24. Friesenecker B, Tsai AG, Intaglietta M. Cellular basis of inflammation, edema and the activity of MPFF at a dose of 500 mg. Int J Microcirc Clin Exp. 1995;15(suppl 1):17-21.
25. Takase S, Lerond L, Bergan JJ, Schmid- Schönbein GW. The inflammatory reaction during venous hypertension in the rat. Microcirculation. 2000;7(1):41-52.
26. Korthuis RJ, Gute DC. Adhesion molecule expression in postischemic microvascular dysfunction: activity of a micronized purified flavonoid fraction. J Vasc Res. 1999;36(suppl 1):15-23. doi:10.1159/000054070
27. de Souza Md, Cyrino FZ, Mayall MR, et al. Beneficial effects of the micronized purified flavonoid fraction (MPFF, MPFF at a dose of 500 mg) on microvascular damage elicited by sclerotherapy. Phlebology. 2016;31(1):50- 56.
28. Katsenis K. Micronized purified flavonoid fraction (MPFF): a review of its pharmacological effects, therapeutic efficacy and benefits in the management of chronic venous insufficiency. Curr Vasc Pharmacol. 2005;3(1):1-9.
29. Allegra C, Bartolo M Jr, Carioti B, Cassiani D. An original microhaemorheological approach to the pharmacological effects of MPFF at a dose of 500 mg in severe chronic venous insufficiency. Int J Microcirc Clin Exp. 1995;15(suppl 1):50-54.
30. Bogachev VY, Boldin BV, Lobanov VN. Benefits of micronized purified flavonoid fraction as adjuvant therapy on inflammatory response after sclerotherapy. Int Angiol. 2018;37(1):71-78.
31. Bogachev VY, Boldin BV, Turkin PY. Administration of micronized purified flavonoid fraction during sclerotherapy of reticular veins and telangiectasias: results of the national, multicenter, observational program VEIN ACT PROLONGED-C1. Adv Ther. 2018;35(7):1001-1008.
32. Pitsch F. Benefit of MPFF at a dose of 500 mg in combination with sclerotherapy of telangiectasias of the lower limbs: results from the SYNERGY and SATISFY surveys. Phlebolymphology. 2011;19(4):182-187.
33. Nicolaides A, Kakkos S, Baekgaard N, et al. Management of chronic venous disorders of the lower limbs. Guidelines According to Scientific Evidence. Part I. Int Angiol. 2018;37(3):181-254.
34. Tsukanov YT, Nikolaichuk AI. Orthostaticloading- induced transient venous refluxes (day orthostatic loading test), and remedial effect of micronized purified flavonoid fraction in patients with telangiectasia and reticular vein. Int Angiol. 2017;36(2):189- 196.
35. Martinez-Zapata MJ, Vernooij RW, Uriona Tuma SM, et al. Phlebotonics for venous insufficiency. Cochrane Database Syst Rev. 2016;4(4):CD003229.
36. Coleridge-Smith P, Lok C, Ramelet A. Venous leg ulcer: a meta-analysis of adjunctive therapy with micronized purified flavonoid fraction. Eur J Vasc Endovasc Surg. 2005;30(2):198-208.
37. Takase S, Bergan JJ, Schmid-Schönbein G. Expression of adhesion molecules and cytokines on saphenous veins in chronic venous insufficiency. Ann Vasc Surg. 2000;14(5):427-435.
38. Gupta N, Zhao YY, Evans CE. The stimulation of thrombosis by hypoxia. Thromb Res. 2019;181:77-83.
39. Smith PC. The causes of skin damage and leg ulceration in chronic venous disease. Int J Low Extrem Wounds. 2006;5(3):160- 168.
40. Tisato V, Zauli G, Rimondi E, et al. Inhibitory effect of natural anti-inflammatory compounds on cytokines released by chronic venous disease patient-derived endothelial cells. Mediators Inflamm. 2013;2013:423407.
41. Castro-Ferreira R, Cardoso R, Leite-Moreira A, Mansilha A. The role of endothelial dysfunction and inflammation in chronic venous disease. Ann Vasc Surg. 2018;46:380-393.
42. MacColl E, Khalil RA. Matrix metalloproteinases as regulators of vein structure and function: implications in chronic venous disease. J Pharmacol Exp Ther. 2015;355(3):410-428.
43. Raffetto JD, Khalil RA. Mechanisms of lower extremity vein dysfunction in chronic venous disease and implications in management of varicose veins. Vessel Plus. 2021;5:36.
44. Lyseng-Williamson KA, Perry CM. Micronised purified flavonoid fraction: a review of its use in chronic venous insufficiency, venous ulcers and haemorrhoids. Drugs. 2003;63(1):71-100.
45. Shoab SS, Porter J, Scurr JH, Coleridge- Smith PD. Endothelial activation response to oral micronised flavonoid therapy in patients with chronic venous disease–a prospective study. Eur J Vasc Endovasc Surg. 1999;17(4):313-318.
46. Shoab SS, Porter JB, Scurr JH, Coleridge- Smith PD. Effect of oral micronized purified flavonoid fraction treatment on leukocyte adhesion molecule expression in patients with chronic venous disease: a pilot study. J Vasc Surg. 2000;31(3):456-461.
47. Boisseau MR. Leukocyte involvement in the signs and symptoms of chronic venous disease. Perspectives for therapy. Clin Hemorheol Microcirc. 2007;37(3):277- 290.
48. Shoab SS, Scurr JH, Coleridge-Smith PD. Plasma VEGF as a marker of therapy in patients with chronic venous disease treated with oral micronised flavonoid fraction – a pilot study. Eur J Vasc Endovasc Surg. 1999;18(4):334-338.
49. Jean T, Bodinier MC. Mediators involved in inflammation: effects of MPFF at a dose of 500 mg on their release. Angiology. 1994;45(6 Pt 2):554-559.
50. Lonchampt M, Guardiola B, Sicot N, Bertrand M, Perdrix L, Duhault J. Protective effect of a purified flavonoid fraction against reactive oxygen radicals. In vivo and in vitro study. Arzneimittelforschung. 1989;39(8):882-885.
51. Juteau N, Bakri F, Pomies JP, et al. The human saphenous vein in pharmacology: effect of a new micronized flavonoidic fraction (MPFF at a dose of 500 mg) on norepinephrine induced contraction. Int Angiol. 1995;14(3 suppl 1):8-13.
52. Gargouil YM, Perdrix L, Chapelain B, Gaborieau R. Effects of MPFF at a dose of 500 mg on bovine vessels contractility. Int Angiol. 1989;8(4 suppl):19-22.
53. Savineau JP, Marthan R. Diosmin-induced increase in sensitivity to Ca2+ of the smooth muscle contractile apparatus in the rat isolated femoral vein. Br J Pharmacol. 1994;111(4):978-980.
54. Tsoukanov YT, Tsoukanov AY, Nikolaychuk A. Great saphenous vein transitory reflux in patients with symptoms related to chronic venous disorders, but without visible signs (C0s), and its correction with MPFF treatment. Phlebolymphology. 2015;85(2):25.