Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jun 1.
Published in final edited form as: J Vasc Surg. 2011 Dec 30;55(6):1716–1725. doi: 10.1016/j.jvs.2011.10.124

Endothelium-Dependent Nitric Oxide and Hyperpolarization-Mediated Venous Relaxation Pathways in Rat Inferior Vena cava

Joseph D Raffetto 1,2,3, Peng Yu 1, Ossama M Reslan 1, Yin Xia 1, Raouf A Khalil 1,2
PMCID: PMC3320697  NIHMSID: NIHMS335792  PMID: 22209615

Abstract

Introduction

The vascular endothelium plays a major role in the control of arterial tone; however, its role in venous tissues is less clear. The purpose of this study was to determine the role of endothelium in the control of venous function, and the relaxation pathways involved.

Methods

Circular segments of inferior vena cava (IVC) from male Sprague-Dawley rats were suspended between two wires and isometric contraction to phenylephrine (Phe, 10−5M) and 96 mM KCl was measured. Acetylcholine (Ach, 10−10 to 10−5M) was added and the percentage venous relaxation was measured. To determine the role of nitric oxide (NO) and prostacyclin (PGI2), vein relaxation was measured in the presence of the NOS inhibitor L-NAME (3X10−4 M) and the COX inhibitor indomethacin (10−5 M). To measure the role of hyperpolarization, vein relaxation was measured in the presence of K+ channel activator cromakalim (10−11 to 10−6 M), and the nonselective K+ channel blocker tetraethylammonium (TEA, 10−3 M). To test for the contribution of a specific K+ channel, the effects of K+ channel blockers: glibenclamide (ATP-sensitive KATP, 10−5M), 4-aminopyridine (4-AP, voltage-dependent Kv, 10−3M), apamin (small conductance Ca2+-dependent SKCa, 10−7M), and iberiotoxin (large conductance Ca2+-dependent BKCa, 10−8M), on Ach-induced relaxation were tested.

Results

Ach caused concentration-dependent relaxation of Phe contraction (max 49.9±4.9%). Removal of endothelium abolished Ach-induced relaxation. IVC treatment with L-NAME partially reduced Ach relaxation (32.8±4.9%). In IVC treated with L-NAME plus indomethacin significant Ach-induced relaxation (33.6±3.2%) could still be observed, suggesting a role of endothelium-derived hyperpolarizing factor (EDHF). In IVC treated with L-NAME, indomethacin and TEA, Ach relaxation was abolished, supporting a role of EDHF. In veins stimulated with high KCl, Ach caused relaxation (max 59.5±3.5%) that was abolished in the presence of L-NAME and indomethacin suggesting that any Ach-induced EDHF is blocked in the presence of high KCl depolarizing solution, which does not favor outward movement of K+ ion and membrane hyperpolarization. Cromakalim, activator of KATP, caused significant IVC relaxation when applied alone or on top of maximal Ach-induced relaxation, suggesting that the Ach response may not involve KATP. Ach-induced relaxation was not inhibited by glibenclamide, 4-AP or apamin, suggesting little role of KATP, Kv or SKCa, respectively. In contrast, iberiotoxin significantly inhibited Ach-induced relaxation, suggesting a role of BKCa.

Conclusions

Thus endothelium-dependent venous relaxation plays a major role in the control of venous function. In addition to NO, an EDHF pathway involving BKCa may play a role in endothelium-dependent venous relaxation.

INTRODUCTION

The vascular endothelium plays an important role in regulating arterial tone, and endothelial dysfunction could lead to progressive arterial disease such as hypertension and atherosclerosis.1–4 An increase in shear stress on the endothelium triggers the release of endothelium-derived substances including nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF).5–10 Endothelial NO diffuses into vascular smooth muscle cells (VSMCs) where it activates guanylate cyclase and the NO-cGMP relaxation pathway, while PGI2 activates adenylate cyclase and the PGI2-cAMP relaxation pathway.11–13 In some arteries particularly the small resistance arteries, blocking the NO-cGMP pathway with the NO synthase inhibitor L-NAME and the PGI2-cAMP pathway with indomethacin does not completely inhibit endothelium-dependent acetylcholine (Ach) relaxation, suggesting the release of EDHF.8, 12, 14 EDHF could cause hyperpolarization of endothelial cells which propagates to VSMCs through myo-endothelial gap junctions, or could cause the release of diffusible factors from the endothelium which act on VSMCs leading to activation of K+ channels, hyperpolarization, and vascular relaxation.1, 8, 15, 16 Several types of K+ channels have been identified in the vasculature including large conductance Ca2+-activated (BKCa), intermediate conductance Ca2+-activated (IKCa), small conductance Ca2+-activated (SKCa), ATP-sensitive (KATP), voltage-gated (KV), and inward rectifier (KIR).8, 16–18 The relative contribution of NO, PGI2 and EDHF to arterial relaxation varies depending on the artery size and specific arterial bed. For instance, NO is a major factor in the relaxation of large arteries such as the aorta.19–21 On the other hand, EDHF plays a major role in relaxation of small resistance arteries such as the mesenteric microvessels.22, 23

Although the pathways of vascular relaxation have been well-characterized in the arterial system, the role of NO-cGMP, PGI2-cAMP, and EDHF in venous relaxation is less defined.24–26 Also, while the majority of K+ channels have been characterized in the arterial system, little is known regarding the K+ channels involved in venous hyperpolarization and relaxation. The aim of this study was to determine the role of the endothelium in the control of venous function, and the potential endothelium-dependent venous relaxation pathways involved.

METHODS

Animals and tissues

Male Sprague-Dawley rats (12 weeks of age, 250–300g in weight, Charles River Laboratories, Wilmington, MA) were maintained on ad libitum standard rat chow and tap water in 12 hr/12 hr light/dark cycle. Rats were euthanized by inhalation of CO2. The inferior vena cava (IVC) was rapidly excised, placed in oxygenated Krebs solution, and carefully dissected and cleaned of connective tissue under microscopic visualization. The IVC was portioned into 3 mm rings in preparation for isometric contraction experiments. From each rat IVC, four 3 mm-wide segments were obtained. All vein segments were obtained from the IVC below the renal veins. Extreme care was taken throughout the tissue isolation and dissection procedure in order to minimize injury to the endothelium and the vein wall. One vein segment from each individual rat was used to perform one experiment and obtain one set of data points. Cumulative data from different vein segments from different rats were used to calculate the average data for each arm of the study. All procedures followed the NIH guide for the Care of Laboratory Animal Welfare Act, and the guidelines of the Animal Care and Use Committee at Harvard Medical School.

Isometric contraction

Circular segments of IVC were suspended between two stainless-steel hooks, one hook was fixed at the bottom of a tissue bath and the other hook was connected to a Grass force displacement transducer (FT03, Astro-Med Inc., West Warwick, RI). Vein segments were stretched under 0.5 g of resting tension and allowed to equilibrate for 45 min in a tissue bath filled with 50 ml Krebs solution continuously bubbled with 95% O2 5% CO2 at 37°C. We have previously constructed the relationship between basal tension and the contraction to 96 mM KCl in rat IVC, and demonstrated that 0.5 g basal tension produced maximal KCl contraction. 27 The changes in isometric contraction/relaxation were recorded on a Grass polygraph (Model 7D, Astro-Med Inc.) as previously described.5

Control IVC contraction in response to 96 mM KCl was first elicited. Once the KCl maximum contraction was reached and a plateau achieved (within 10 to 15 min) the tissue was washed 3 times in Krebs, 10 min each. The control contraction to 96 mM KCl was repeated twice prior to further experimentation.

To investigate the venous endothelial function, IVC segments were precontracted with phenylephrine (Phe, 10−5 M) then treated with increasing concentrations (10−10 to 10−5 M) of Ach and the % venous relaxation was measured. To test the role of the endothelium, Ach-induced relaxation was compared in intact and endothelium-denuded IVC. In these experiments, the endothelium was mechanically removed by gently scraping the intimal surface of the IVC segment five times around the tip of forceps.5 To test the role of endothelium-dependent NO-cGMP relaxation pathway, experiments were repeated in IVC segments treated with the NO synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 3x10−4 M), and the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo[4,2-a]quinoxalin-1-one (ODQ, 10−5 M). To inhibit cyclooxygenase (COX) the veins were pretreated for 10 min with indomethacin (10−5 M). To test the role of hyperpolarization and potential release of EDHF, the effects of Ach (10−5 M) were tested on vein contraction induced by membrane depolarization by 96 mM KCl. To test for the potential K+ channel involved in hyperpolarization, the effects of Ach on Phe-induced IVC contraction were compared with those of the K+ channel opener and KATP activator cromakalim (10−11 to 10−6 M). To further test for the specific K+ channel involved, Ach-induced relaxation of Phe contraction was examined in veins pretreated with the following K+ channel blockers: non-selective blocker of Ca2+-dependent K+ channel tetraethylammonium (TEA, 10−3 M), KATP blocker glibenclamide (10−5 M), KV blocker 4-aminopyridine (4-AP, 10−3 M), SKCa blocker apamin (10−7 M), and BKCa blocker iberiotoxin (10−8 M).3, 5–8, 16, 28 The concentrations of inhibitors of NOS, COX, and K+ channels were determined on the basis of previous studies and published IC50.

Solutions, Drugs and Chemicals

Normal Krebs solution contained in mM: NaCl 120, KCl 5.9, NaHCO3 25, NaH2PO4 1.2, dextrose 11.5 (Fisher Scientific, Fair Lawn, NJ), CaCl2 2.5 (BDH Laboratory Supplies Poole, England), and MgCl2 1.2 (Sigma, St. Louis, MO). Krebs solution was bubbled with 95% O2 and 5% CO2 for 30 min, at an adjusted pH 7.4. 96 mM KCl was prepared as normal Krebs but with equimolar substitution of NaCl with KCl. Stock solutions of Phe (10−1 M), Ach (10−1 M), Nω-L-nitro-arginine methyl ester (L-NAME, 10−1 M), 4-AP (10−1 M), apamin (10−3 M) (Sigma), and iberiotoxin (10−5 M) (Calbiochem, La Jolla, CA) were prepared in distilled water. TEA (Sigma) was prepared as 1 mM solution in Krebs. Stock solutions of 1H-(1,2,4)oxadiazolo[4,2-a]quinoxalin-1-one (ODQ, 10−1) (Calbiochem), indomethacin (10−1 M), cromakalim (10−2 M), and glibenclamide (10−1 M) (Sigma) were prepared in diemtheylsulfoxide (DMSO). The final concentration of DMSO in the experimental solution was <0.1%. All other chemicals were of reagent grade or better.

Statistical Analysis

Cumulative data were analyzed and presented as means±SEM with the “n” value representing the number of experiments on different vein segments from 4 to 12 rats. Data were compared using Student’s t-test for unpaired data and differences were considered statistically significant if P < 0.05.

RESULTS

Ach-Induced Endothelium-Dependent Relaxation

In endothelium-intact IVC, Phe (10−5 M) caused significant contraction (0.07±0.01 g/mg tissue weight) that was maintained at steady-state for approximately 20 min. Topical application of increasing concentrations of Ach (10−9 to 10−5 M), caused concentration dependent relaxation of Phe contraction that reached a maximum of 49.9±4.9% at 10−5 M. In endothelium-denuded IVC, Ach relaxation was completely absent (0.2±0.2%, P<0.001), indicating that the Ach-induced IVC relaxation is endothelium-dependent (Fig. 1).

Fig. 1.

Fig. 1

Open in a new tab

Ach-induced relaxation in rat IVC. Endothelium intact IVC segments were precontracted with Phe (10−5 M), increasing Ach concentrations were added, the vein relaxation was observed (A), and the % relaxation of Phe contraction was measured (B). In other experiments, the effects of Ach were measured in endothelium-denuded IVC (B). Data represent means±SEM (n=12 to 31). * Significant (p < 0.05).

Role of NO-cGMP

Pretreatment of IVC segments with the NOS blocker L-NAME (3x10−4 M) caused a significant decrease in Ach-induced relaxation to a maximum of 32.8±4.9% (P<0.01) at 10−5 M. Also, pretreatment of IVC segments with the guanylate cyclase inhibitor ODQ (10−5 M) caused significant reduction of Ach relaxation to a maximum of 16.7±5.3% (P<0.001) at 10−5 M (Fig. 2). These data indicated a role for NO-cGMP pathway in IVC relaxation. However, Ach-induced relaxation was not completely inhibited by either L-NAME or ODQ, suggesting the involvement of other venous relaxation pathways.

Fig. 2.

Fig. 2

Open in a new tab

Role of NO-cGMP in Ach-induced relaxation of rat IVC. IVC segments were either nontreated or pretreated with L-NAME (3x10−4 M) or ODQ (10−5 M) for 15 min. IVC segments were then precontracted with Phe (10−5 M), increasing Ach concentrations were added, and the % relaxation of Phe contraction was measured. Data represent means±SEM (n=8 to 31). * Significant (p < 0.05).

Contribution of PGI2 and EDHF

To evaluate the role of PGI2 in the Ach-induced relaxation observed during NO blockade by L-NAME, veins were treated with the COX inhibitor indomethacin (INDO, 10−5 M). Compared to control Ach-induced relaxation (49.9±4.9%), in IVC treated with both L-NAME+INDO, Ach caused significantly less relaxation that reached a maximum of 33.6±3.2% at 10−5 M (Fig. 3). Ach-induced relaxation in the presence of L-NAME+INDO was not significantly different from that in the presence of L-NAME alone, suggesting little role of PGI2. In IVC treated with L-NAME+INDO+non-selective Ca2+ dependent K+ channel blocker TEA, Ach-induced relaxation was abolished (0.5±0.3%), suggesting a role of EDHF (Fig. 3).

Fig. 3.

Fig. 3

Open in a new tab

Contribution of NO, PGI2, and EDHF to Ach relaxation of rat IVC. IVC segments were either nontreated or pretreated with L-NAME (3x10−4 M) plus indomethacin (INDO, 10−5 M) or L-NAME+INDO+tetraethylammonium (TEA, 10−3 M) for 15 min. IVC segments were then precontracted with Phe (10−5 M), increasing Ach concentrations were added, and the % relaxation of Phe contraction was measured. The L-NAME-sensitive component of Ach relaxation (~35%) is attributed to NO, while the TEA-sensitive component (~65%) is attributed to EDHF. Data represent means±SEM (n=16 to 31). * Significant (p < 0.05).

Effect of Ach on KCl-induced contraction

High 96 mM KCl depolarizing solution creates a K+ gradient that does not favor outward movement of K+ ion and hyperpolarization.25, 29 KCl caused significant IVC contraction (0.27±0.02 g/mg tissue weight) that was maintained at steady-state for approximately 20 min. Ach caused concentration-dependent relaxation of KCl-induced contraction that reached a maximum of 59.5±3.5% at 10−5 M. In IVC treated with L-NAME+INDO, Ach-induced relaxation of KCl contraction was significantly reduced (5.7±1.0%, P<0.0001) (Fig. 4). The absence of Ach-induced relaxation of KCl contraction in IVC treated with L-NAME+INDO suggests that any Ach-induced EDHF and hyperpolarization is blocked in the presence of high KCl depolarizing solution.

Fig. 4.

Fig. 4

Open in a new tab

Contribution of NO, PGI2, and EDHF to Ach relaxation of depolarization-induced contraction of rat IVC. IVC segments were either nontreated or pretreated with L-NAME (3x10−4 M) plus indomethacin (INDO, 10−5 M) for 15 min. IVC segments were then precontracted with high KCl (96 mM) depolarizing solution, increasing Ach concentrations were added, and the % relaxation of KCl contraction was measured. The L-NAME-sensitive component of Ach relaxation of KCl contraction (~95%) is attributed to NO, and almost no relaxation is attributed to EDHF. Data represent means±SEM (n=12). * Significant (p < 0.05).

Effect of KATP Channel Opener Cromakalim

The prototype KATP channel activator cromakalim caused concentration-dependent relaxation of Phe contraction that reached a maximum of 91.0±1.6% at 10−6 M (Fig. 5A). In contrast, with Ach-induced relaxation of KCl contraction, cromakalim caused minimal relaxation of KCl-induced relaxation (3.1±1.3% at 10−6 M) (Fig. 5B), supporting that hyperpolarization is an important relaxation mechanism in IVC segments.

Fig. 5.

Fig. 5

Open in a new tab

Contribution of hyperpolarization pathway to relaxation of rat IVC. IVC segments were precontracted with either Phe (10−5 M) (A) or high KCl (96 mM) depolarizing solution (B). IVC segments were then treated with increasing concentrations of the KATP channel activator cromakalim, or Ach for comparison. Compared to the Ach response, cromakalim caused larger relaxation of Phe-induced contraction, but almost no relaxation of depolarization-induced KCl contraction. Data represent means±SEM (n=8 to 31).

Interaction between NO-Dependent and Hyperpolarization-Dependent Vein Relaxation

In IVC treated with L-NAME+INDO, Ach still caused significant relaxation of Phe contraction, and addition of cromakalim (10−6 M) caused further relaxation of Phe contraction to 58.8±5.3%, P<0.001) (Fig. 6). Because cromakalim activates KATP, its additive effects to maximal Ach-induced relaxation suggest that Ach may activate K+ channels other than KATP. In comparison, in IVC treated with L-NAME+INDO, cromakalim caused relaxation of Phe contraction (61.0±4.6%), but was unexpectedly less than the relaxation caused by cromakalim alone (91.0±1.6%), suggesting cross talk between the NO-cGMP and hyperpolarization pathways. On the other hand, Ach did not cause any further significant relaxation of Phe-contraction in veins treated with cromakalim in the presence of L-NAME+INDO (Fig. 6), likely due to cromakalim-induced maximal hyperpolarization.

Fig. 6.

Fig. 6

Open in a new tab

Interaction between NO-dependent and hyperpolarization-dependent relaxation in rat IVC. IVC segments were precontracted with Phe (10−5 M) and a control Ach (10−5 M) or cromakalim (10−6 M) relaxation was elicited (open bars). In parallel experiments, IVC segments were pretreated with L-NAME (3x10−4 M)+indomethacin (INDO, 10−5 M) for 15 min, precontracted with Phe then treated with Ach (10−5 M) or cromakalim (10−6 M) (dotted bars). At steady-state, the veins treated with Ach were further challenged with cromakalim, while the veins treated with cromakalim were further challenged with Ach, and the veins were observed for any further relaxation (Solid bars). Cromakalim caused further relaxation of Ach-treated veins, while Ach did not cause any further significant relaxation of cromakalim-treated veins. Data represent means±SEM (n=7 to 31).

* Measurements in L-NAME+INDO treated veins are significantly different (p<0.05) from corresponding measurements in control veins.

# Measurements in veins treated with L-NAME+INDO+Ach followed by cromakalim are significantly different (p<0.05) from corresponding measurements in control veins.

Effect of Selective K+ Channel Blockers

Ach could utilize one or more K+ channel to produce IVC hyperpolarization.8, 16–18 In order to determine the K+ channel(s) involved in the IVC relaxation, selective K+ channels blockers were utilized. Compared to control Ach-induced relaxation, Ach-induced relaxation was not significantly reduced in IVC treated with KATP blocker glibenclamide, Kv blocker 4-AP, or SKCa blocker apamin. In contrast, Ach-induced relaxation was significantly reduced in IVC treated with the BKCa blocker IbTx (Fig. 7).

Fig. 7.

Fig. 7

Open in a new tab

Effect of selective K+ channel blockers on Ach relaxation of rat IVC. IVC segments were precontracted with Phe (10−5 M) and a control Ach (10−5 M)-induced relaxation was elicited. At steady-state the veins were treated with a specific K+ channel blocker: KATP blocker glibenclamide (10−5 M), KV blocker 4-aminopyridine (4-AP, 10−3 M), SKCa blocker apamin (10−7 M), and BKCa blocker iberiotoxin (IbTX, 10−8 M), and the remaining Ach relaxation was measured. Data represent means±SEM (n=4 to 31).

* Measurements in veins treated with K+ channel blocker are significantly different (p<0.05) from corresponding measurements in control veins.

DISCUSSION

The present findings in rat IVC demonstrate that: 1) Ach causes significant endothelium-dependent venous relaxation. 2) Approximately one-third of Ach-induced venous relaxation involves NO synthesis and the NO-cGMP pathway. 3) Hyperpolarization is an important mechanism of venous relaxation. 4) Approximately two-thirds of Ach-induced venous relaxation involves activation of BKCa-dependent hyperpolarization pathway. 5) There is a cross talk between the hyperpolarization-dependent and NO-cGMP pathways during vein relaxation.

Several groups have examined endothelium-dependent relaxation in numerous arterial preparations.30–33. However, our knowledge regarding the endothelium-dependent venous relaxation pathways is limited.24–26 This is in part because the venous tissue is very delicate and difficult to handle. We have previously shown that the rat IVC produces significant and measurable contraction.5, 34 Consistent with our previous reports, we found that the rat IVC produced significant Phe contraction. The Phe contraction was maintained for at least 20 min, making it possible to measure Ach-induced vein relaxation.

Consistent with previous reports in arterial segments,35, 36 Ach-induced IVC relaxation was concentration-dependent and reached a maximum at 10−5 M concentration. Also, the magnitude of Ach-induced venous relaxation (50–60%) was in the range of Ach-induced relaxation in arterial segments. Ach-induced venous relaxation was endothelium-dependent because it was nearly abolished in endothelium-denuded veins. Our results are consistent with an earlier study demonstrating endothelium-dependent relaxation in rat femoral veins.25 These observations suggest that the venous endothelium is functional and produces significant relaxation of venous tissue.

The endothelium is known to release several vasodilator substances including NO, PGI2, and EDHF.5–8 Endothelial-derived vasodilators are released in response to shear stress acting on endothelial cell, and also in response to neurohumoral mediators in the circulation acting on specific receptors on the endothelium including growth factors, cathecholamines, histamine, Ach, arachidonic acid, bradykinin, serotonin, thrombin, endothelin, arginine vasopressin, and adenosine diphosphate released by aggregating platelets.8 In large blood vessels such as the aorta, pharmacologic inhibitors of NOS and COX almost abolish Ach-induced relaxation. In contrast, in small resistance arteries pretreated with NOS and COX inhibitors, a significant component of Ach-induced relaxation is still observed, suggesting a role of EDHF in the regulation of arterial tone.1, 8

Endothelium-derived NO is known to diffuse into VSM where it activates guanylate cyclase and increases cGMP production. cGMP activates cGMP-dependent protein kinase (PKG) which promotes Ca2+ extrusion and decreases VSM [Ca2+]i and also causes phosphorylation and inactivation of myosin light chain kinase leading to inhibition of VSM contraction and consequent vascular relaxation.11, 13 We have previously shown that the rat IVC produces significant amounts of NO under basal conditions, and that Ach produces significant increases in NO production.5 Consistent with our previous report, we found that Ach-induced IVC relaxation was partly blocked by the NOS inhibitor L-NAME. Also, inhibition of guanylate cyclase by ODQ caused significant decrease in Ach-induced relaxation, indicating the importance of NO-cGMP pathway in venous relaxation.1, 37, 38 However, a significant Ach-induced relaxation could still be observed in L-NAME treated IVC, suggesting the involvement of other EDRF(s). Pretreatment of IVC with L-NAME plus the COX inhibitor INDO did not cause any further reduction in Ach-induced relaxation, suggesting little role of PGI2. This study in rat IVC demonstrated that one-third of Ach relaxation was due to NO, and by excluding PGI2, the remaining two-thirds of Ach-induced relaxation appear to involve EDHF. One way to block EDHF is to use high KCl depolarizing solution, which creates a K+ gradient that does not favor outward movement of K+ ion and therefore prevents hyperpolarization.25, 29 Ach caused approximately 60% relaxation of KCl contraction, that was abolished in IVC treated with L-NAME+INDO, further supporting a role of the NO-cGMP pathways. Importantly, in IVC treated with both L-NAME and INDO, Ach still caused relaxation of Phe contraction, but failed to cause relaxation of KCl contraction. These data support a role of EDHF in Ach-induced relaxation, and that this hyperpolarization factor is blocked in the presence of KCl depolarizing solution. These data also support that EDHF is an important pathway in not only the regulation of arterial tone,25, 26 but also the regulation of venous function.

Although the exact nature of the EDHF molecule(s) is not exactly known, several candidate compounds have been studied and likely function as EDRF and lead to hyperpolarization. Potassium ions are known to accumulate in the intercellular space between endothelial and smooth muscle cells. The potassium ions form what is called a potassium cloud that activates SKCa and IKCa on the endothelium and hyperpolarization ensues by activating both KIR and Na+/K+ pump on the SMC.10 Other important EDHFs are metabolites of arachidonic acid produced by cytochrome P450 monooxygenase and are called epoxyeicosatrienoic acid (EET).39 EETs are known to hyperpolarize smooth muscle by activating BKCa and other K+ channels.40 Other EDHF candidates are hydrogen peroxide that may activate K+ channels on the endothelium or SMC,15 but the mechanism is unclear. Additionally, C-type natriuretic peptide leads to hyperpolarization of both arterial and venous tissue by activating BKCa.8, 26

The current study was not intended to characterize the EDHF molecule, but to highlight the role of hyperpolarization and potential K+ channel(s) involved in IVC relaxation. There are several K+ channel openers that include cromakalim, pinacidil, nicorandil, diazoxide and RP-49356.29 Cromakalim works mainly by activating KATP channels.41 Our data demonstrate significant IVC relaxation in response to cromakalim, supporting a role of hyperpolarization and activation of KATP channels as a major mechanism of IVC relaxation. This is consistent with other studies demonstrating a significant relaxation to cromakalim in human saphenous vein.42, 43 Importantly, the venorelaxant effect of cromakalim was essentially abolished in IVC pre-contracted with 96mM KCl. High KCl solution abrogates the outward flow of K+ from the endothelium and SMC via K+ channels, and hence completely eliminates the possibility for hyperpolarization, and hence relaxation. Our findings are consistent with previous reports in both rat and human venous tissues.25, 42

In search for the potential K+ channel involved in IVC relaxation, we found that Ach-induced relaxation was not reversed in the presence of specific blockers of KATP, KV, or SKCa. In contrast, Ach-induced IVC relaxation was abolished in the presence of the BKCa blocker iberiotoxin. These findings suggest that a key modulator of hyperpolarization in the IVC venous system is via the activation of BKCa channel. Our data are consistent with reports that human umbilical vein endothelial cells express BKCa.44 Thus while in the arterial system EDHF may require the activation of SKCa and also IKCa to cause hyperpolarization, 3, 8, 45, 46 this may not be the situation in veins, particularly the IVC.

NO not only activates guanylate cyclase to produce cGMP, but can also co-activate various K+ channels including KATP, KV, BKCa.1, 8 In porcine internal mammary and rat mesenteric arteries, Ach-induced relaxation via the NO-cGMP pathway could also involve activation of SKCa, IKCa and KV channels.37, 47 We found that cromakalim caused less relaxation in the presence of L-NAME and INDO than cromakalim alone. This would indicate that NO, either directly or indirectly via increasing cGMP, is an important effector molecule in enhancing KATP channel activity during hyperpolarization and relaxation of rat IVC (Fig. 8).

Fig. 8.

Fig. 8

Open in a new tab

Venous relaxation pathways in rat IVC. Ach stimulates the NO-cGMP pathway to cause vein relaxation. Ach also activates BKCa leading to hyperpolarization and further vein relaxation. Cromakalim activates KATP causing hyperpolarization and vein relaxation. NO-cGMP could directly or indirectly enhance KATP activity via a cross-talk mechanism.

It is important to note that the present study was conducted on rat IVC. Although the rat IVC is a very thin and delicate preparation, we have perfected the vein harvesting and dissection technique and obtained reproducible data and consistent findings.5, 27, 48, 49 Our tissue histology studies demonstrate that similar to human veins, the rat IVC has three anatomical layers; intima, media and adventitia.34 Also, physiological vein function studies demonstrate that the rat IVC contract and relax to various constrictor and vasodilator stimuli.5, 49 Although the rat is a four-legged animal, studies on rat tissues avoid variability in age, body weight and other confounding factors in humans. To enhance the relevance of the present findings to lower extremity veins, studies should be conducted on rat iliac and femoral vein. However, because human lower extremity veins are subjected to more pressure in the upright posture and may adapt differently to pressure/stretch, one needs to be careful in extrapolating the present data in rat veins to human veins. We have previously studied vein functions in circular segments of human saphenous vein and varicose veins.50 We have shown that membrane depolarization by high 96 mM KCl solution causes significant contraction of human saphenous vein. Whether membrane hyperpolarization plays a role in human vein relaxation is unclear and should represent an important area for future investigations. Furthermore, to enhance the translational aspects, future studies should characterize not only the relaxation pathways in control greater saphenous vein but also the changes in the venous relaxation mechanisms in varicose veins.

Venous wall disease could take several forms ranging from vein restenosis and graft failure 51 to venous dilation and varicose veins.52 Dilated varicose veins, both primary and recurrent, are known to involve valve dysfunction with increased venous pressure leading to wall dilation and refluxing valves in any segment of the superficial and deep system.53 However, venous wall dilation could also precede valve dysfunction and may have an equally important role in the pathophysiology of varicose vein development.52, 54. The identification of the mechanisms underlying venous relaxation could be important in understanding the pathophysiological mechanisms underlying the vein wall stenosis associated with graft failure and vein wall dilation associated with varicose vein formation. Targeting these abnormal venodilator mechanisms could be useful in the management of venous disease. Pharmacological activators of the NO pathway and K+ channels could be useful in reducing vein restenosis and graft failure. On the other hand, pharmacologic therapy utilizing specific inhibitors of the NO pathway and specific K+ channel blockers could be useful in the management of primary and recurrent varicose veins.

Vein valvular disease has been suggested to cause vein reflux and subsequent venous dilation. However, venous wall duplex ultrasonographic studies have described an ascending phenomenon to the pathophysiology of varicose veins, suggesting that vein wall dilation may precede valvular dysfunction.52 Also, biochemical abnormalities similar to those observed in varicose veins have been demonstrated in normal-appearing human saphenous vein wall adjacent to varicose veins and between competent venous valves.55, 56 We have also shown that prolonged increases in vein wall tension in rat IVC is associated with venous dysfunction and decreased vein contraction.27 The increased vein wall tension causes an increase in matrix metalloproteinases MMP-2 and MMP-9, and MMPs in turn cause vein wall hyperpolarization and venous relaxation possibly by activating large conductance Ca2+ dependent K+ channels.5. MMP-2 induced hyperpolarization could cause inhibition of Ca2+ entry into venous smooth muscle.34 The present study supports the contention that hyperpolarization is a major relaxation pathway in veins. We postulate that persistent and recalcitrant venous hypertension leads to increased vein wall tension and release of MMP-2 and MMP-9, which in turn cause hyperpolarization and inhibition of Ca2+ entry into venous smooth muscle and lead to decreased contraction and the increased venous dilation associated with varicose veins.27 It is likely that both valve dysfunction and vein wall dilation play a role in the pathophysiology of varicose veins. Thus while removing reflux would lead to remodeling and “shrink down” of varicose veins in humans, it would also remove the persistent venous hypertension and the increased vein wall tension. This will in turn reverse the increase in MMPs, and the persistent vein wall hyperpolarization and the venous dilation associated with varicose veins.

In conclusion, the present study elucidated some of the mechanisms involved in relaxation of rat IVC. Importantly, venous relaxation involves endothelium-dependent pathways that are mediated by both NO-cGMP and EDHF, with an important contribution of hyperpolarization via BKCa and KATP channels. Further research will be required to determine the role of NO-cGMP, PGI2-cAMP, and EDHF relaxation pathways in human saphenous veins and varicose veins, and to examine potential pharmacologic interventions that can be useful in the treatment of the over-distended varicose veins.

CLINICAL RELEVANCE.

Endothelial dysfunction plays a major role in the pathogenesis of arterial disease, and could also affect the course of venous disease. Although the endothelium-derived mediators have been well characterized in the arterial wall, the mechanisms of venous dilation are poorly understood. The present study in rat inferior vena cava demonstrates that the mechanisms of venous relaxation partly involve the NO pathway, as well as a significant portion involving the hyperpolarization pathway. While activation of ATP-sensitive K+ channels (KATP) could cause venous relaxation, Ach-induced endothelium-dependent relaxation appears to involve the large conductance Ca2+-dependent K+ channel (BKCa). The identification of the mechanisms of venous relaxation could be important in the management of venous disease. Pharmacological activators of the NO pathway and K+ channels could be useful in reducing vein restenosis and graft failure. On the other hand, pharmacologic therapy utilizing specific blockers of the NO pathway and K+ channels could be useful in the management of primary and recurrent varicose veins.

Acknowledgments

R. A. Khalil was partly supported by grants from the National Heart, Lung, and Blood Institute (HL-65998, HL-98724), and The Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD-60702).

List of Non-Standard Abbreviations

cAMP

cyclic adenosine monophosphate

cGMP

cyclic guanosine monophosphate

COX

cyclooxygenase

EDHF

endothelium-derived hyperpolarizing factor

EDRF

endothelium-derived relaxing factor

NO

nitric oxide

NOS

NO synthase

PGI2

prostacyclin

Phe

phenylephrine

VSM

vascular smooth muscle

References

  • 1.Feletou M, Kohler R, Vanhoutte PM. Endothelium-derived vasoactive factors and hypertension: possible roles in pathogenesis and as treatment targets. Curr Hypertens Rep. 2010;12(4):267–275. doi: 10.1007/s11906-010-0118-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Vanhoutte PM, Shimokawa H, Tang EH, Feletou M. Endothelial dysfunction and vascular disease. Acta Physiol (Oxf) 2009;196(2):193–222. doi: 10.1111/j.1748-1716.2009.01964.x. [DOI] [PubMed] [Google Scholar]
  • 3.Grgic I, Kaistha BP, Hoyer J, Kohler R. Endothelial Ca+-activated K+ channels in normal and impaired EDHF-dilator responses--relevance to cardiovascular pathologies and drug discovery. Br J Pharmacol. 2009;157(4):509–526. doi: 10.1111/j.1476-5381.2009.00132.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Vanhoutte PM. Endothelial dysfunction: the first step toward coronary arteriosclerosis. Circ J. 2009;73(4):595–601. doi: 10.1253/circj.cj-08-1169. [DOI] [PubMed] [Google Scholar]
  • 5.Raffetto JD, Ross RL, Khalil RA. Matrix metalloproteinase 2-induced venous dilation via hyperpolarization and activation of K+ channels: relevance to varicose vein formation. J Vasc Surg. 2007;45(2):373–380. doi: 10.1016/j.jvs.2006.10.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ross GR, Yallampalli C. Endothelium-independent relaxation by adrenomedullin in pregnant rat mesenteric artery: role of cAMP-dependent protein kinase A and calcium-activated potassium channels. J Pharmacol Exp Ther. 2006;317(3):1269–1275. doi: 10.1124/jpet.106.101790. [DOI] [PubMed] [Google Scholar]
  • 7.Kohler R, Hoyer J. The endothelium-derived hyperpolarizing factor: insights from genetic animal models. Kidney Int. 2007;72(2):145–150. doi: 10.1038/sj.ki.5002303. [DOI] [PubMed] [Google Scholar]
  • 8.Feletou M, Vanhoutte PM. Endothelium-derived hyperpolarizing factor: where are we now? Arterioscler Thromb Vasc Biol. 2006;26(6):1215–1225. doi: 10.1161/01.ATV.0000217611.81085.c5. [DOI] [PubMed] [Google Scholar]
  • 9.Brandes RP, Schmitz-Winnenthal FH, Feletou M, Godecke A, Huang PL, Vanhoutte PM, Fleming I, Busse R. An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice. Proc Natl Acad Sci U S A. 2000;97(17):9747–9752. doi: 10.1073/pnas.97.17.9747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Edwards G, Dora KA, Gardener MJ, Garland CJ, Weston AH. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature. 1998;396(6708):269–272. doi: 10.1038/24388. [DOI] [PubMed] [Google Scholar]
  • 11.Fleming I, Busse R. NO: the primary EDRF. J Mol Cell Cardiol. 1999;31(1):5–14. doi: 10.1006/jmcc.1998.0839. [DOI] [PubMed] [Google Scholar]
  • 12.Parkington HC, Coleman HA, Tare M. Prostacyclin and endothelium-dependent hyperpolarization. Pharmacol Res. 2004;49(6):509–514. doi: 10.1016/j.phrs.2003.11.012. [DOI] [PubMed] [Google Scholar]
  • 13.Ignarro LJ, Kadowitz PJ. The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation. Annu Rev Pharmacol Toxicol. 1985;25:171–191. doi: 10.1146/annurev.pa.25.040185.001131. [DOI] [PubMed] [Google Scholar]
  • 14.Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, Weston AH. EDHF: bringing the concepts together. Trends Pharmacol Sci. 2002;23(8):374–380. doi: 10.1016/s0165-6147(02)02050-3. [DOI] [PubMed] [Google Scholar]
  • 15.Shimokawa H, Morikawa K. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in animals and humans. J Mol Cell Cardiol. 2005;39(5):725–732. doi: 10.1016/j.yjmcc.2005.07.007. [DOI] [PubMed] [Google Scholar]
  • 16.Edwards G, Feletou M, Weston AH. Endothelium-derived hyperpolarising factors and associated pathways: a synopsis. Pflugers Arch. 2010;459(6):863–879. doi: 10.1007/s00424-010-0817-1. [DOI] [PubMed] [Google Scholar]
  • 17.Coleman HA, Tare M, Parkington HC. Endothelial potassium channels, endothelium-dependent hyperpolarization and the regulation of vascular tone in health and disease. Clin Exp Pharmacol Physiol. 2004;31(9):641–649. doi: 10.1111/j.1440-1681.2004.04053.x. [DOI] [PubMed] [Google Scholar]
  • 18.Zygmunt PM, Edwards G, Weston AH, Larsson B, Hogestatt ED. Involvement of voltage-dependent potassium channels in the EDHF-mediated relaxation of rat hepatic artery. Br J Pharmacol. 1997;121(1):141–149. doi: 10.1038/sj.bjp.0701108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Al-Zobaidy MJ, Craig J, Martin W. Differential sensitivity of basal and acetylcholine-induced activity of nitric oxide to blockade by asymmetric dimethylarginine in the rat aorta. Br J Pharmacol. 2010;160(6):1476–1483. doi: 10.1111/j.1476-5381.2010.00802.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Zembowicz A, Hatchett RJ, Jakubowski AM, Gryglewski RJ. Involvement of nitric oxide in the endothelium-dependent relaxation induced by hydrogen peroxide in the rabbit aorta. Br J Pharmacol. 1993;110(1):151–158. doi: 10.1111/j.1476-5381.1993.tb13785.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Davis JR, Giardina JB, Green GM, Alexander BT, Granger JP, Khalil RA. Reduced endothelial NO-cGMP vascular relaxation pathway during TNF-alpha-induced hypertension in pregnant rats. Am J Physiol Regul Integr Comp Physiol. 2002;282(2):R390–399. doi: 10.1152/ajpregu.00270.2001. [DOI] [PubMed] [Google Scholar]
  • 22.Gerber RT, Anwar MA, Poston L. Enhanced acetylcholine induced relaxation in small mesenteric arteries from pregnant rats: an important role for endothelium-derived hyperpolarizing factor (EDHF) Br J Pharmacol. 1998;125(3):455–460. doi: 10.1038/sj.bjp.0702099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Brondum E, Kold-Petersen H, Simonsen U, Aalkjaer C. NS309 restores EDHF-type relaxation in mesenteric small arteries from type 2 diabetic ZDF rats. Br J Pharmacol. 2010;159(1):154–165. doi: 10.1111/j.1476-5381.2009.00525.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Komori K, Shimokawa H, Vanhoutte PM. Endothelium-dependent relaxation in response to aggregating platelets in porcine femoral veins and its modulation by diet. Circulation. 1989;80(2):401–409. doi: 10.1161/01.cir.80.2.401. [DOI] [PubMed] [Google Scholar]
  • 25.Nagao T, Vanhoutte PM. Hyperpolarization contributes to endothelium-dependent relaxations to acetylcholine in femoral veins of rats. Am J Physiol. 1991;261(4 Pt 2):H1034–1037. doi: 10.1152/ajpheart.1991.261.4.H1034. [DOI] [PubMed] [Google Scholar]
  • 26.Banks M, Wei CM, Kim CH, Burnett JC, Jr, Miller VM. Mechanism of relaxations to C-type natriuretic peptide in veins. Am J Physiol. 1996;271(5 Pt 2):H1907–1911. doi: 10.1152/ajpheart.1996.271.5.H1907. [DOI] [PubMed] [Google Scholar]
  • 27.Raffetto JD, Qiao X, Koledova VV, Khalil RA. Prolonged increases in vein wall tension increase matrix metalloproteinases and decrease constriction in rat vena cava: Potential implications in varicose veins. J Vasc Surg. 2008;48(2):447–456. doi: 10.1016/j.jvs.2008.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kitamura K, Kamouchi M. K channel openers activate different K channels in vascular smooth muscle cells. Cardiovasc Drugs Ther. 1993;7 (Suppl 3):539–546. doi: 10.1007/BF00877619. [DOI] [PubMed] [Google Scholar]
  • 29.Weston AH, Longmore J, Newgreen DT, Edwards G, Bray KM, Duty S. The potassium channel openers: a new class of vasorelaxants. Blood Vessels. 1990;27(2–5):306–313. doi: 10.1159/000158823. [DOI] [PubMed] [Google Scholar]
  • 30.Ng KF, Leung SW, Man RY, Vanhoutte PM. Endothelium-derived hyperpolarizing factor mediated relaxations in pig coronary arteries do not involve Gi/o proteins. Acta Pharmacol Sin. 2008;29(12):1419–1424. doi: 10.1111/j.1745-7254.2008.00905.x. [DOI] [PubMed] [Google Scholar]
  • 31.Shi Y, Ku DD, Man RY, Vanhoutte PM. Augmented endothelium-derived hyperpolarizing factor-mediated relaxations attenuate endothelial dysfunction in femoral and mesenteric, but not in carotid arteries from type I diabetic rats. J Pharmacol Exp Ther. 2006;318(1):276–281. doi: 10.1124/jpet.105.099739. [DOI] [PubMed] [Google Scholar]
  • 32.Zellers TM, Wu YQ, McCormick J, Vanhoutte PM. Prostacyclin-induced relaxations of small porcine pulmonary arteries are enhanced by the basal release of endothelium-derived nitric oxide through an effect on cyclic GMP-inhibited-cyclic AMP phosphodiesterase. Acta Pharmacol Sin. 2000;21(2):131–138. [PubMed] [Google Scholar]
  • 33.Bussemaker E, Popp R, Binder J, Busse R, Fleming I. Characterization of the endothelium-derived hyperpolarizing factor (EDHF) response in the human interlobar artery. Kidney Int. 2003;63(5):1749–1755. doi: 10.1046/j.1523-1755.2003.00910.x. [DOI] [PubMed] [Google Scholar]
  • 34.Raffetto JD, Barros YV, Wells AK, Khalil RA. MMP-2 induced vein relaxation via inhibition of [Ca2+]e-dependent mechanisms of venous smooth muscle contraction. Role of RGD peptides. J Surg Res. 2010;159(2):755–764. doi: 10.1016/j.jss.2008.09.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Gauthier KM, Goldman DH, Aggarwal NT, Chawengsub Y, Falck JR, Campbell WB. Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries. Am J Physiol Heart Circ Physiol. 2011;300(3):H725–735. doi: 10.1152/ajpheart.00696.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Lu X, Kassab GS. Assessment of endothelial function of large, medium, and small vessels: a unified myograph. Am J Physiol Heart Circ Physiol. 2011;300(1):H94–H100. doi: 10.1152/ajpheart.00708.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Favaloro JL, Kemp-Harper BK. Redox variants of NO (NO{middle dot} and HNO) elicit vasorelaxation of resistance arteries via distinct mechanisms. Am J Physiol Heart Circ Physiol. 2009;296(5):H1274–1280. doi: 10.1152/ajpheart.00008.2009. [DOI] [PubMed] [Google Scholar]
  • 38.Pimentel AM, Costa CA, Carvalho LC, Brandao RM, Rangel BM, Tano T, Soares de Moura R, Resende AC. The role of NO-cGMP pathway and potassium channels on the relaxation induced by clonidine in the rat mesenteric arterial bed. Vascul Pharmacol. 2007;46(5):353–359. doi: 10.1016/j.vph.2006.12.003. [DOI] [PubMed] [Google Scholar]
  • 39.Fleming I, Busse R. Endothelium-derived epoxyeicosatrienoic acids and vascular function. Hypertension. 2006;47(4):629–633. doi: 10.1161/01.HYP.0000208597.87957.89. [DOI] [PubMed] [Google Scholar]
  • 40.McSherry IN, Sandow SL, Campbell WB, Falck JR, Hill MA, Dora KA. A role for heterocellular coupling and EETs in dilation of rat cremaster arteries. Microcirculation. 2006;13(2):119–130. doi: 10.1080/10739680500466400. [DOI] [PubMed] [Google Scholar]
  • 41.Glavind-Kristensen M, Matchkov V, Hansen VB, Forman A, Nilsson H, Aalkjaer C. KATP-channel-induced vasodilation is modulated by the Na,K-pump activity in rabbit coronary small arteries. Br J Pharmacol. 2004;143(7):872–880. doi: 10.1038/sj.bjp.0706016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.de Moura RS, Jasbik W. Actions of cromakalim in isolated human saphenous vein. Hypertension. 1992;19(2 Suppl):II121–124. doi: 10.1161/01.hyp.19.2_suppl.ii121. [DOI] [PubMed] [Google Scholar]
  • 43.Beattie DK, Gosling M, Davies AH, Powell JT. The effects of potassium channel openers on saphenous vein exposed to arterial flow. Eur J Vasc Endovasc Surg. 1998;15(3):244–249. doi: 10.1016/s1078-5884(98)80184-6. [DOI] [PubMed] [Google Scholar]
  • 44.Kohler R, Schonfelder G, Hopp H, Distler A, Hoyer J. Stretch-activated cation channel in human umbilical vein endothelium in normal pregnancy and in preeclampsia. J Hypertens. 1998;16(8):1149–1156. doi: 10.1097/00004872-199816080-00011. [DOI] [PubMed] [Google Scholar]
  • 45.Eichler I, Wibawa J, Grgic I, Knorr A, Brakemeier S, Pries AR, Hoyer J, Kohler R. Selective blockade of endothelial Ca2+-activated small- and intermediate-conductance K+-channels suppresses EDHF-mediated vasodilation. Br J Pharmacol. 2003;138(4):594–601. doi: 10.1038/sj.bjp.0705075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Kohler R, Wulff H, Eichler I, Kneifel M, Neumann D, Knorr A, Grgic I, Kampfe D, Si H, Wibawa J, Real R, Borner K, Brakemeier S, Orzechowski HD, Reusch HP, Paul M, Chandy KG, Hoyer J. Blockade of the intermediate-conductance calcium-activated potassium channel as a new therapeutic strategy for restenosis. Circulation. 2003;108(9):1119–1125. doi: 10.1161/01.CIR.0000086464.04719.DD. [DOI] [PubMed] [Google Scholar]
  • 47.Pagan RM, Prieto D, Hernandez M, Correa C, Garcia-Sacristan A, Benedito S, Martinez AC. Regulation of NO-dependent acetylcholine relaxation by K+ channels and the Na+-K+ ATPase pump in porcine internal mammary artery. Eur J Pharmacol. 2010;641(1):61–66. doi: 10.1016/j.ejphar.2010.05.004. [DOI] [PubMed] [Google Scholar]
  • 48.Raffetto JD, Khalil RA. Ca(2+)-dependent contraction by the saponoside escin in rat vena cava: implications in venotonic treatment of varicose veins. J Vasc Surg. 2011;54(2):489–496. doi: 10.1016/j.jvs.2011.01.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Raffetto JD, Qiao X, Beauregard KG, Khalil RA. Estrogen receptor-mediated enhancement of venous relaxation in female rat: implications in sex-related differences in varicose veins. J Vasc Surg. 2010;51(4):972–981. doi: 10.1016/j.jvs.2009.11.074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Raffetto JD, Qiao X, Beauregard KG, Tanbe AF, Kumar A, Mam V, Khalil RA. Functional adaptation of venous smooth muscle response to vasoconstriction in proximal, distal, and varix segments of varicose veins. J Vasc Surg. 2010;51(4):962–971. doi: 10.1016/j.jvs.2009.11.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Conte MS. Technical factors in lower-extremity vein bypass surgery: how can we improve outcomes? Semin Vasc Surg. 2009;22(4):227–233. doi: 10.1053/j.semvascsurg.2009.10.004. [DOI] [PubMed] [Google Scholar]
  • 52.Raffetto JD, Khalil RA. Mechanisms of varicose vein formation: valve dysfunction and wall dilation. Phlebology. 2008;23(2):85–98. doi: 10.1258/phleb.2007.007027. [DOI] [PubMed] [Google Scholar]
  • 53.Labropoulos N, Giannoukas AD, Delis K, Mansour MA, Kang SS, Nicolaides AN, Lumley J, Baker WH. Where does venous reflux start? J Vasc Surg. 1997;26(5):736–742. doi: 10.1016/s0741-5214(97)70084-3. [DOI] [PubMed] [Google Scholar]
  • 54.Bergan JJ, Schmid-Schonbein GW, Smith PD, Nicolaides AN, Boisseau MR, Eklof B. Chronic venous disease. N Engl J Med. 2006;355(5):488–498. doi: 10.1056/NEJMra055289. [DOI] [PubMed] [Google Scholar]
  • 55.Kirsch D, Dienes HP, Kuchle R, Duschner H, Wahl W, Bottger T, Junginger T. Changes in the extracellular matrix of the vein wall--the cause of primary varicosis? Vasa. 2000;29(3):173–177. doi: 10.1024/0301-1526.29.3.173. [DOI] [PubMed] [Google Scholar]
  • 56.Kirsch D, Wahl W, Bottger T, Junginger T. Primary varicose veins--changes in the venous wall and elastic behavior. Chirurg. 2000;71(3):300–305. doi: 10.1007/s001040050049. discussion 305–306. [DOI] [PubMed] [Google Scholar]

RESOURCES