Comparative histopathological study of the venous wall of chronic venous insufficiency and varicose disease

Abstract

Objectivew

To investigate venous histopathology of chronic venous insufficiency and varicose patients (C2).

Methods

Retrospective review of venous histopathology of 52 patients (13, 8, 2, and 28 were C2, C4, C5 and C6).

Results

The intimal thickness, intimal fibrosis, total thickness and intimal/total thickness ratio were highest in venous clinical severity score 0, 1 chronic venous insufficiency (no or minimal varicosity) follow by Venous Clinical Severity Score 2,3 chronic venous insufficiency (trunkal varice) and C2 veins (mean intimal thickness 62, 36, 26 µm, mean intimal fibrosis 74%, 72%, 65%, mean total thickness 184, 159, 133 µm, mean intimal/total thickness ratio 0.32, 0.20, 0.21). The statistical significances were found when comparing intimal thickness, intimal fibrosis, intimal/total thickness ratio and total thickness of Venous Clinical Severity Score 0, 1 chronic venous insufficiency veins and C2 veins. The medial changes are relatively constant among groups.

Conclusion

Compared with C2 vein, the intimal changes in chronic venous insufficiency venous wall differ, particularly in the VCSS 0, 1 chronic venous insufficiency.

Keywords

CVI chronic venous insufficiency histopathology histology

Introduction

Chronic venous insufficiency (CVI), an advanced form of chronic venous disease with skin change (clinical severity class 4 or C4), healed (C5) or active venous ulceration (C6), is a common but under-recognized disease in Asia. Between 3.6 and 18%^1–3 of the western population suffers from CVI (CEAP classification C4–6) and 0.1–2.6%^1,2,4,5 are affected by venous ulcers (CEAP classification C5–6). The exact prevalence of CVI in Asia is not known but may be substantial. Patients with skin change or ulceration constituted 1.5% of our general surgery admission and 32% of patients presented with venous disease in a Hong Kong survey.⁶ Traditionally, CVI which is characterized by hyperpigmentation, liposclerosis and ulceration is thought to be the continuous spectrum of advanced varicose vein disease. Our previous report documented that, even in the patients with advanced stage of CVI with venous ulceration (C6), varicosity is infrequent.⁷ We hypothesized that though advanced varicose disease may account for a certain percentage of CVI patients, a considerable proportion may be related to other undetermined nature of venous reflux. This study aims to investigate the venous wall histopathology of CVI patients with no or minimal varicosity (VCSS 0 or 1); CVI patients with conspicuous varicosity (VCSS 2 or 3); and patients with varicose disease patients without skin changes (C2).

Material and methods

Venous specimens from successive CVI (C4-6) or varicose (C2) patients who had undergone great saphenous and small saphenous vein stripping at the surgical department, Vajira Hospital between 1 January 2006 and 31 August 2013 were prospectively collected and retrospectively reviewed.

Preoperatively, the standard protocol was used for collecting relevant clinical history and findings. Clinical assessment of the severity of varicosity in each patient was assessed using the venous clinical severity score (VCSS),⁸ which categorized varicose severity into: grade 0: none; grade 1: few scattered branches; grade 2: multiple great saphenous vein confined to calf or thigh and grade 3: extensive thigh and calf of great saphenous or small saphenous distribution. The reflux of the great or small saphenous vein was validated by preoperative ultrasonography. The technique of ultrasonographic examination was described elsewhere.⁷ Reflux time of more than 500 ms was defined as venous reflux. The histological specimens from CVI legs were dichotomized into two groups: those which were removed from the legs with varicose VCSS grades 0 and 1, and those which were removed from the legs with varicose VCSS grades 2 and 3.

For the bilateral specimens of the same patient, only the randomly selected side was included to the study. Venous specimens from the patients with history of deep vein thrombosis or ultrasonographic evidence of venous thrombosis or obstruction were excluded.

The venous specimens were preserved in 10% neutral buffered formalin solution. The CEAP classification of the subjects was blinded to the pathologist. The 5 µm thick paraffin specimens of three random sections along venous specimens (proximal, middle and distal) were prepared and stained with Hematoxylin & Eosin and Masson's trichrome. The stained slides were examined under light microscope for evaluating general histopathological change and measuring into quantitative scale. In each specimen, the thickness of venous wall layers was calculated from the mean values of measurements at three random locations. Collagen fiber deposition of the intimal and media layers was quantified from the proportion of Masson’s trichrome stained area over non-stained area represented as percentage values using image analyzer program (MeeSoft Image analyzer program, version 1.36.1). The data were analyzed using descriptive statistics. The Mann-Whitney U test was used for the comparison of the means. The SPSS-9 (Chicago, IL, USA) software was used for statistical analysis. The Faculty of Medicine, Vajira Hospital Ethical Committee approved this study.

Results

The venous specimens were collected from 51 patients; 18 and 33 were male and female, respectively. The mean age of the CVI patients (C4–6) was significantly higher than the mean age of the varicose patients (C2) (60.4 ± 12.2 year (27–82 year) versus 53.0 ± 9.3 year (30–64 year), (p = 0.38)). Thirteen, 8, 2 and 28 patients were categorized as C2, C4, C5 and C6, respectively. In the 38 CVI patients, there was predominance (28 patients, 74%) of patients with active venous ulceration (C6). The majority of CVI patients had no or minimal varicosity (VCSS 0 or 1) (25 patients, 66%), while a moderate to severe degree of varicosity (VCSS 2 or 3) was presented in 13 patients (34%). The specimens comprised 48 great saphenous veins and three small saphenous veins. The general characteristics the studied population were summarized in Table 1.

Table 1.

General characteristics of the patients.

	All patients n = 51	Varicose disease (C2) n = 13	CVI (C4–6) n = 38
Age (year)	58.2 ± 11.9	53.0 ± 9.3	60.4 ± 12.2
	(30–82)	(30–64)	(27–82)
Sex (male:female)	18:33	2:11	16:22
Side of venous specimens (left:right:bilateral)	30:18:3	8:4:1	22:13:2
CEAP classification	–	C2, n = 13	C4, n = 8, C5, n = 2, C6, n = 28
VCSS of visible varicosity	–	VCSS 2, n = 4	VCSS 0 n = 19
		VCSS 3, n = 11	VCSS 1 n = 6
			VCSS 2 n = 7
			VCSS 3 n = 6

VCSS: venous clinical severity score; CVI: chronic venous insufficiency.

Comparing the specimens of the CVI (C4-6) patients versus the varicose patients (C2) (Table 2, comparison 1). The intimal thickness and percent fibrosis of the CVI patients were significantly higher than the varicose patients (C2) (intimal thickness, mean 52.9 µm ± 41.4 (5.3–149.6) versus mean 25.9 µm ± 15.9 (6.4–59.0), p = .015, intimal fibrosis, mean 73% ± 9 (56–91) versus 65% ± 10 (50–82), p = .015).

Table 2.

Comparative histological finding between groups.

Comparisons	Intima thickness (µm)	Media thickness (µm)	Total wall thickness (µm)	Intima/total wall thickness ratio	Intima fibrosis (%)	Media fibrosis (%)
1. All CVI (C4–C6) (n = 38) versus	52.9 ± 41.4	122.9 ± 50.3	175.8 ± 80.3	0.28 ± 0.13	73 ± 9	57 ± 10
1. All CVI (C4–C6) (n = 38) versus	(5.3–149.6)	(50.2–246.9)	(68.6–371.1)	(0.07–0.57)	(56–91)	(36–81)
Varicose patients (C2) (n = 13)	25.9 ± 15.9	106.7 ± 48.7	132.6 ± 48.1	0.21 ± 0.13	65 ± 10	59 ± 11
Varicose patients (C2) (n = 13)	(6.4–59.0)	(31.0–205.1)	(50.2–266.1)	(0.05–0.49)	(50–82)	(41–74)
Statistical significance	p = .015 (Sig)	p = .342	p = .110	p = .101	p = .015 (Sig)	p = .523
2. CVI with no or minimal varicosity (VCSS 0,1) (n = 25) versus	61.7 ± 41.3	122.8 ± 51.2	184.5 ± 81.3	0.32 ± 0.13	74 ± 10	58 ± 9
	(5.3–144.0)	(50.2–246.9)	(71.1–371.1)	(0.07–0.57)	(56–91)	(44–81)
Varicose patients (C2) (n = 13)	25.9 ± 15.9	106.7 ± 48.7	132.6 ± 48.1	0.21 ± 0.13	65 ± 10	59 ± 11
Varicose patients (C2) (n = 13)	(6.4–59.0)	(31.0–205.1)	(50.2–266.1)	(0.05–0.49)	(50–82)	(41–74)
Statistical significance	p = .002 (Sig)	p = .361	p = .056	p = .023 (Sig)	p = .023 (Sig)	p = .879
3. CVI with moderate or severe varicosity (VCSS 2,3) (n = 13) versus	36.0 ± 37.5	123.0 ± 50 6	159.0 ± 78.6	0.20 ± 0.10	72 ± 7	53 ± 10
	(6.9–149.6)	(56.4–232.2)	(68.6–322.7)	(0.09–0.46)	(62–88)	(36–72)
Varicose patients (C2) (n = 13)	25.9 ± 15.9	106.7 ± 48.7	132.6 ± 48.1	0.21 ± 0.13	65 ± 10	59 ± 11
Varicose patients (C2) (n = 13)	(6.4–59.0)	(31.0–205.1)	(50.2–266.1)	(0.05–0.49)	(50–82)	(41–74)
Statistical significance	p = .418	p = .511	p = 0.579	p = 1	p = .044 (Sig)	p = .223
4. CVI with no or minimal varicosity (VCSS 0,1) (n = 25) versus	61.7 ± 41.3	122.8 ± 51.2	184.5 ± 81.3	0.32 ± 0.13	74 ± 10	58 ± 9
	(5.3–144.0)	(50.2–246.9)	(71.1–371.1)	(0.07–0.57)	(56–91)	(44–81)
CVI with moderate or severe varicosity (VCSS 2,3) (n = 13)	36.0 ± 37.5	123.0 ± 50.6	159.0 ± 78.6	0.20 ± 0.10	72 ± 7	53 ± 10
CVI with moderate or severe varicosity (VCSS 2,3) (n = 13)	(6.9–149.6)	(56.4–232.2)	(68.6–322.7)	(0.09–0.46)	(62–88)	(36–72)
Statistical significance	p = .028 (Sig)	p = .952	p = .272	p = .006 (Sig)	p = .649	p = .125
5. CVI age ≥ 60.4 (n = 20) versus	46.1 ± 33.2	120.3 ± 49.3	166.3 ± 70.9	0.26 ± 0.12	75 ± 10	60 ± 9
5. CVI age ≥ 60.4 (n = 20) versus	(5.3–130.5)	(50.1–232.2)	(71.0–318.7)	(0.07–0.49)	(57–91)	(44–81)
CVI age < 60.4 (n = 18)	60.6 ± 49.0	125.7 ± 52.7	186.3 ± 90.5	0.29 ± 0.14	70 ± 8	53 ± 9
CVI age < 60.4 (n = 18)	(6.9–149.6)	(51.2–246.9)	(68.6–371.1)	(0.09–0.57)	(56–88)	(36–68)
Statistical significance	p = .443	p = .762	p = .613	p = .654	p = .099	p = .013 (Sig)
6. CVI with no or minimal varicosity (VCSS 0,1) age ≤ 64 (n = 16) versus	75.8 ± 44.2	130.1 ± 55.2	205.9 ± 87.7	0.35 ± 0.13	73 ± 11	57 ± 8
	(5.3–144.0)	(51.2–247.0)	(77.6–371.1)	(0.07–0.57)	(56–91)	(41–69)
Varicose patients (C2) (n = 13)	25.9 ± 15.9	106.7 ± 48.7	132.6 ± 48.1	0.21 ± 0.13	65 ± 10	59 ± 11
Varicose patients (C2) (n = 13)	(6.4–59.0)	(31.0–205.1)	(50.2–266.1)	(0.05–0.49)	(50–82)	(41–74)
Statistical significance	p < .001 (Sig)	p = .249	p = .020 (Sig)	p = .012 (Sig)	p = .040 (Sig)	p = .589
7. Female CVI with no or minimal varicosity (VCSS 0,1) (n = 16) versus	74.4 ± 45.0	132.5 ± 49.6	206.8 ± 84.6	0.34 ± 0.12	74 ± 10	60 ± 10
	(13.3–143.9)	(63.5–246.9)	(92.8–371.1)	(0.14–0.57)	(57–91)	(41–81)
Female Varicose patients (C2) (n = 11)	26.3 ± 17.3	109.0 ± 52.6	135.2 ± 51.7	0.22 ± 0.14	66 ± 10	58 ± 11
Female Varicose patients (C2) (n = 11)	(6.4–59.0)	(31.0–205.1)	(50.2–266.1)	(0.05–0.49)	(50–82)	(41–74)
Statistical significance	p = .001(Sig)	p = .272	p = .034 (Sig)	p = .026 (Sig)	p = .056	p = .680
8. Male CVI (n = 16) versus	32.5 ± 17.7	111.5 ± 45.6	144.1 ± 51.7	0.23 ± 0.12	73 ± 9	56 ± 10
8. Male CVI (n = 16) versus	(5.3–74.3)	(50.2–206.5)	(71.0–246.3)	(0.07–0.50)	(56–88)	(36–72)
Female CVI (n = 22)	67.8 ± 47.5	131.1 ± 53.0	198.8 ± 90.1	0.31 ± 0.13	73 ± 9	57 ± 10
Female CVI (n = 22)	(6.9–149.6)	(56.4–246.9)	(68.6–371.1)	(0.09–0.57)	(57–91)	(41–81)
Statistical significance	p = .024 (Sig)	p = .298	p = .060	p = .064	p = .849	p = .715

VCSS: venous clinical severity score; CVI: chronic venous insufficiency.

Comparing the specimens from the minimal or no varicosity CVI patients (VCSS 0 or 1) with the varicose patients (C2) (Table 2, comparison 2), the intimal thickness, intimal/total wall thickness ratio and the percent intimal fibrosis of the specimens from the VCSS 0 or 1 CVI patients were significantly higher than the varicose patients (C2) (intimal thickness, mean 61.7 µm ± 41.3 (5.3–144.0) versus mean 25.9 µm ± 15.9 (6.4–59.0), p = .002, intimal/total wall thickness ratio, mean 0.32 ± 0.13 (0.07–0.57) versus 0.21 ± 0.13 (0.05–0.49), p = .023, percent intimal fibrosis mean 74% ± 10 (56–91) versus 65% ± 10 (50–82), p = .023)

Comparing the specimens from the moderate or severe varicosity CVI patients (VCSS 2 or 3) with the varicose patients (C2) (Table 2, comparison 3), only the percent intimal fibrosis of the VCSS 2 or 3 CVI patients was significantly higher (percent intimal fibrosis mean 72% ± 7 (62–88) versus 65% ± 10 (50–82), p = .044)).

Comparing the specimens from the absent or minimal varicosity CVI patients (VCSS 0 or 1) with the moderate or severe varicosity CVI patients (VCSS 2 or 3) (Table 2, comparison 4), the intimal thickness and the intimal/total wall thickness ratio of the VCSS 0 or 1 CVI patients were significantly higher than the specimens from the VCSS 2 or 3 CVI patients (intimal thickness, mean 61.7 µm ± 41.3 (5.3–144.0) versus mean 36.0 µm ± 37.5 (6.9–149.6), p = .028, intimal/total wall thickness ratio mean 0.32 ± 0.13 (0.07–0.57) versus 0.20 ± 0.10 (0.09–0.46), p = .006).

There were no statistical differences among the medial thickness and medial fibrosis among all of the above comparisons.

Comparing the specimens of the older CVI patients (age>60.4 years, n = 20) with the younger CVI patients (n = 18) (Table 2, comparison 5), only the comparison of medial fibrosis was statistically significant (mean 60 ± 9 (44–81) versus, 53 ± 9 (36–68) p = .013). The specimens of the CVI patients with no or minimal varicosity (VCSS 0 or 1) age between 25 and64 years were (n = 16) were sorted out and compared with the varicose patients (C2, n = 13 age, range 27–64 years)) (Table 2, comparison 6), the intimal thickness, total wall thickness, intimal/total wall thickness ratio and intimal fibrosis of the specimens of the VCSS 0 or 1 CVI patients were significantly higher than the specimens of the varicose patients (C2) (intimal thickness, mean 75.8 µm ± 44.2 (5.3–144.0) versus mean 25.9 µm ± 15.9 (6.4–59.0), p < .001, total wall thickness, mean 205.9 ± 87.7 (77.6–371.1) versus 132.6 ± 48.1 (50.2–266.1) p = .020, intimal/total wall thickness ratio mean 0.35 ± 0.13 (0.07–0.57) versus 0.21 ± 0.13(0.05–0.49), p = .012, percent intimal fibrosis mean 73% ± 11 (56–91) versus 65% ± 10 (50–82), p = .040).

Comparing the specimens of the VCSS 0 or 1 female CVI patients with the female varicose patients (C2) (Table 2, comparison 7), the intimal thickness, total wall thickness and intimal/total wall thickness ratio of the female VCSS 0 or 1 CVI patients were significantly higher (intimal thickness, mean 74.4 µm ± 45.0 (13.3–143.9) versus mean 26.3 µm ± 17.3 (6.4–59.0), p = .001, total wall thickness, mean 206.8 µm ± 84.6 (92.8–371.1) versus mean 135.2 µm ± 51.7 (50.2–266.1) p = .034, intimal/total wall thickness ratio mean 0.34 ± 0.12 (0.14–0.57) versus mean 0.22 ± 0.14 (0.05–0.49) p = .026. When comparing the specimens of the male with the female CVI patients (Table 2, comparison 8), only the difference in intimal thickness was statistically significant (mean 32.5 ± 17.7 (5.3–74.3) versus 67.8 ± 47.5 (6.9–149.6) p = .024).

Discussion

Our previous study revealed that the visible varicosity (VCSS 1-3) is present in approximately half of the CVI legs (45%) and when present, it tends to be mild.⁷ The observation that the majority of CVI patients do not harbor varicosity was also observed by Welch et al.⁹ who reported observable varicose veins in only 46% of patients with venous ulceration (C6).

The histopathological findings of varicose venous walls are well described. These include intimal hypertrophy, venous wall fibrosis and disruption of the elastin and smooth muscle cells organization.^10–12 The loss of structural scaffold mass and integrity such as elastic fragmentation and reduced amount of extracellular matrix is believed to lead to structural weakness, dilatation and venous reflux.^10–12 However, very few studies described the histologic features of the C4–6 CVI venous wall.¹³

This study recognized that the histological features of the venous wall of CVI patients differ from the varicose patients. While the wall thickening and fibrosis are found in both CVI and varicose venous walls, the abnormalities in CVI venous wall are more severe. The differences seem to be most prominent in intimal layer as evidenced by intense intimal thickening, overall wall thickening, increase in intimal/total wall thickening ratio and fibrosis. Moreover, the histologic abnormalities are more intense and diffuse in venous wall of CVI legs with no or minimal observable varicosity. This group (VCSS 0,1) has been shown to constitute the largest proportion of CVI population.⁷The finding in this study, combined with the fact that the majority of varicose patients, although advanced, did not have skin change or ulceration characteristic of CVI, challenges the commonly quoted concept that the CVI evolve as a continuous progressive state of varicose disease.^10,11

The higher prevalence of CVI in elderly population has been documented by many previous studies.^2,4,5,7 Non-appearance of statistical difference in any parameters other than medial fibrosis when comparing the specimens from younger CVI patients to the older CVI patients foreclose the possibility that the difference between CVI and varicose patients was confounded by age. In addition, when the specimens from the VCSS 0 or 1 CVI patients with the age that matched the age range of C2 patients were compared with C2 group, the differences of the intimal layer histopathology between two groups were reconfirmed. To confirm that the results were not biased by the higher proportional of female sex in the varicose patients, the specimens from the female VCSS 0 or 1 CVI patients were compared with the specimens from the female varicose patients; the differences of intimal layer histopathology were verified. In addition, the specimens from male CVI patients were compared with the specimens from the female CVI patients. Except for intimal thickness, no statistical difference in any others parameters was found.

Although CVI is common, insight in to the pathogenesis is limited. Increased inflammatory mediators and growth factors such as intercellular adhesion molecule (ICAM 1), von Willebrand factor, transforming growth factor β₁ (TGF-β₁), fibroblast growth factor β (FGF-β) and matrix metalloproteinases (MMPs)^11,12,14 have been shown to increase in varicose venous walls, which suggests the interaction between venous hypertension and inflammation in the wall structural changes. Further study of these substances in the CVI venous wall may be worthwhile.

Limitation of this study included possible bias from large proportion of the patients with advance disease (C6). The retrospective nature of the study precluded comparison of specific location along saphenous veins among groups. Small number of population, especially in subgroup analysis emphasizes the necessary for further research. Our limited experience suggests that the C3 (leg edema) patients have the same feature of no or minimal visible varicosity similar to the C4–6 patients in this study. These were not included due to paucity of the C3 patients, probably related to the patient’s less likelihood to seeks medical service, the difficulty to related the non-specific symptoms to the venous disease and less inclination for surgical management in the patients with less severe disease.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Faculty of Medicine, Vajira Hospital research fund funded this research.

References

Rabe

Pannier

. What we learned from the Bonn vein study. Phlebolymphology 2006; 13: 188–194.

Coon

Willis

Keller

. Venous thromboembolism and other venous disease in the Tecumseh Community Health Study. Circulation 1973; 48: 839–846.

Chiesa

Marone

Limoni

. Demographic factors and their relationship with the presence of CVI signs in Italy: the 24-cities cohort study. Eur J Vasc Endovasc Surg 2005; 30: 674–680.

Cornwall

Dore

Lewis

. Leg ulcers: epidemiology and aetiology. Br J Surg 1986; 73: 1032–1034.

Cesarone

Belcaro

Nicolaides

. ‘Real’ epidemiology of varicose veins and chronic venous diseases: the San Valentino Vascular Screening Project. Angiology 2002; 53: 119–130.

Ting

ACW

Cheng

SWK

. Surgical treatment for advanced chronic venous insufficiency in Hong Kong. Ann Coll Surg Hong Kong 2004; 8: 135–140.

Kanchanabat

Wongmahisorn

Stapanavatr

. Clinical presentation and patterns of venous reflux in Thai patients with chronic venous insufficiency (CVI). Eur J Vasc Endovasc Surg 2010; 40: 399–402.

Rutherford

Padberg

Jr Comerota

. Venous severity scoring: an adjunct to venous outcome assessment. J Vasc Surg 2000; 31: 1307–1312.

Welch

Young

Semegran

. Duplex assessment of venous reflux and chronic venous insufficiency: the significance of deep venous reflux. J Vasc Surg 1996; 24: 755–762.

10.

Lim

Davies

. Pathogenesis of primary varicose veins. BJS 2009; 96: 1231–1242.

11.

Oklu

Habito

Mayr

. Pathogenesis of varicose veins. J Vasc Interv Radiol 2012; 23: 33–39.

12.

Bergan

JJ1

Schmid-Schönbein

Smith

. Chronic venous disease. N Engl J Med 2006; 355: 488–498.

13.

Mironiuc

Palcău

Rogojan

. Is there a correlation between the CEAP score and the histopathological findings in varicose disease? Rom J Morphol Embryol 2011; 52: 117–121.

14.

Nicolaides

. Chronic venous disease and the leukocyte-endothelium interaction: from symptoms to ulceration. Angiology 2005; 56: S11–S19.