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. Author manuscript; available in PMC: 2007 Feb 8.
Published in final edited form as: J Vasc Surg. 2007 Feb;45(2):373–380. doi: 10.1016/j.jvs.2006.10.041

Matrix Metalloproteinase-2 Induced Venous Dilation via Hyperpolarization and Activation of K+ Channels. Relevance to Varicose Vein Formation

Joseph D Raffetto 3, Reagan L Ross 1, Raouf A Khalil 1,2
PMCID: PMC1794684  NIHMSID: NIHMS13550  PMID: 17264019

Abstract

Background

Varicose veins are a common disorder of extensive venous dilation and remodeling with as yet unclear mechanism. Studies have shown elevated plasma and tissue levels of matrix metalloproteinases (MMPs) in human varicose veins and animal models of venous hypertension. Although the effects of MMPs are generally attributed to extracellular matrix degradation, their effects on the mechanisms of venous contraction/relaxation are unclear. Our preliminary experiments have demonstrated that MMP-2 causes inhibition of phenylephrine (Phe)-induced venous contraction. The purpose of this study was to determine whether MMP-induced inhibition of venous contraction involves an endothelium-dependent and/or -independent pathway.

Methods

Circular segments of the inferior vena cava (IVC) were isolated from male Sprague-Dawley rats and suspended between two wire hooks in a tissue bath, and the effects of MMP-2 on Phe- and KCl-induced contraction were measured. To study the role of endothelium-derived vasodilators, experiments were performed in the presence and absence of endothelium; L-NAME, inhibitor of nitric oxide (NO) synthesis; indomethacin, inhibitor of prostacyclin (PGI2) synthesis; cromakalim, activator of ATP-sensitive K+ channel (KATP); and iberiotoxin, blocker of large conductance Ca2+-dependent K+ channel (BKCa) and smooth muscle hyperpolarization.

Results

In endothelium-intact IVC segments, Phe (10−5 mol/L) caused significant contraction that slowly declined to 82.0% in 30 min. Addition of MMP-2 (1 μg/mL) caused a gradual decrease of Phe contraction to 39.5% at 30 min. In endothelium-denuded IVC MMP-2 induced greater reduction of Phe contraction to 7.6%. In presence of L-NAME (10−4 mol/L), MMP-2 caused marked decrease in Phe contraction to 4.4%. Large MMP-2 induced inhibition of Phe contraction was also observed in IVC treated with L-NAME plus indomethacin. MMP-2 caused relaxation of Phe contraction in IVC pretreated with cromakalim (10−7 mol/L), activator of KATP channel. MMP-2 induced inhibition of Phe contraction was abrogated in the presence of iberiotoxin (10−8 mol/L), blocker of BKCa. MMP-2 did not inhibit venous contraction during membrane depolarization by 96 mmol/L KCl, a condition that prevents outward K+ conductance and cell hyperpolarization.

Conclusions

MMP-2 causes significant IVC relaxation that is potentiated in the absence of endothelium or during blockade of endothelium-mediated NO and PGI2 synthesis. The lack of effects of MMP-2 on KCl contraction and in iberiotoxin treated veins suggests MMP-2 induced smooth muscle hyperpolarization and activation of BKCa channel, a novel effect of MMP that may play a role in the early stages of venous dilation and varicose vein formation.

Introduction

Varicose veins is a common disorder with an estimated prevalence between 5% to 30% of the adult population.1 More than 50% of women and 20% of men with varicose veins will have chronic venous insufficiency with clinical manifestations of leg pain and swelling.2 Progressive venous insufficiency can lead to venous ulceration affecting up to 2.5 million people each year.3 Although the risk factors for varicose veins such as female gender, pregnancy, obesity, aging, and family history have been identified,2,4 the molecular mechanisms underlying the pathogenesis and progression of varicose veins remain unclear.

Matrix metalloproteinases (MMPs) are a family of zinc-containing proteolytic enzymes, which degrade and remodel the extracellular matrix.5 MMPs play a key role in wound healing, inflammation, and smooth muscle cell migration.5,6 MMPs also have a significant effect on vascular tissue remodeling that could play a role in the pathogenesis and progression of vascular disease. For example, MMPs have been implicated in the pathogenesis of abdominal aortic aneurysm by inducing destruction of the elastic lamellae and extracellular matrix, arterial wall degradation and weakening, and progressive aortic dilation.7-9 MMPs may also be involved in atherosclerotic plaque instability, and a role of MMP-2 in atherosclerosis and plaque rupture has been demonstrated during arterial lesion progression.10,11

Previous studies have shown that the plasma and venous tissue levels of MMP-1, -2, -3, -9, and -13 are elevated in human varicose veins, suggesting that MMPs may contribute to the pathogenesis of the disease.12-15 MMPs have been detected in all histologic layers of the venous wall, and increased MMP expression and activity have been demonstrated in thrombophlebitic varicose veins.14 However, the presence of MMPs in varicose veins could be merely a component of the chronic inflammatory process. Also, increased MMP activity in the advanced thrombophlebitc stages does not address whether MMPs affect venous function at the early stages of varicose vein formation.

We have previously shown that MMPs cause relaxation of rat aorta and suggested a role of MMPs in the aortic dilation associated with aneurysm formation.16 Also, our preliminary experiments suggest that MMP-2 causes relaxation of phenylephrine contraction in rat inferior vena cava.17 The purpose of this study was to test whether MMP-2 induced venous relaxation involves an endothelium-dependent mechanism, or an endothelium-independent inhibition of venous smooth muscle contraction. To test this novel hypothesis, we evaluated the effects of MMP-2 in rat inferior vena cava in the presence and absence of endothelium, in the presence and absence of inhibitors of endothelium-derived vasodilators, and during modulation of K+ channel activity by depolarizing solutions and by activators and blockers of K+ channels.

Material and Methods

Solutions and drugs

Krebs solution contained (in mmol/L): NaCl 120, KCl 5.9, NaHCO3 25, NaH2PO4 1.2, dextrose 11.5, CaCl2 2.5, MgCl2 1.2, at pH 7.4, and bubbled with 95% O2 and 5% CO2. 96 mol/L KCl was prepared as Krebs solution with equimolar substitution of NaCl with KCl. Tested drugs included Phe, acetylcholine (Ach), N(G)-L-nitro-arginine methyl ester (L-NAME), indomethacin (Sigma, St. Louis, MO), MMP-2 (recombinant human, active form, Biomol, Plymouth Meeting, PA), cromakalim (activator of KATP, Sigma), iberiotoxin (blocker of BKCa, Calbiochem, La Jolla, CA). All other chemicals were of reagent grade or better.

Animals and tissues

Male Sprague-Dawley rats (12 weeks, 250-300 g, Charles River Lab., Wilmington, MA) were euthanized by inhalation of CO2. The abdominal cavity was opened, and 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. All procedures followed the guidelines of the Institutional Animal Care and Use Committee.

Isometric contraction

IVC segments were suspended between two wire hooks, one hook is fixed and the other hook is connected to a Grass force transducer (FT03). Vein segments were stretched under 0.5 g of resting tension and allowed to equilibrate for 45 min in a temperature controlled, water-jacketed tissue bath, filled with 50 ml Krebs solution continuously bubbled with 95% O2 5% CO2 at 37°C. The changes in isometric contraction were recorded on a Grass polygraph (Model 7D, Astro-Med).

Two control 96 mmol/L KCl contractions, each followed by 3 times washing in Krebs solution, were first performed. A contraction to Phe 10−5 mol/L was then elicited. When Phe-induced contraction reached steady-state, MMP-2 was added and its effect was observed. We have previously performed dose-response and time course experiments with MMPs in rat aorta rings.16 We found that MMP-2 at 1 μg/mL concentration caused maximum aortic relaxation in 30 min. Also, our preliminary data on the IVC have shown time-dependent MMP-2 induced relaxation that reached steady-state in 30 min.17 Therefore, in all experiments 1 μg/mL MMP-2 was used and its effects were measured after 30 min. To inhibit NO synthesis or cyclooxygenases the tissues were treated for 10 min with L-NAME (10−4 mol/L) or indomethacin (10−5 mol/L), respectively. The role of K+ channels was tested by measuring the effects of MMP-2 on Phe contraction in the presence of the KATP activator cromakalim (10−7 mol/L) and the BKCa blocker iberiotoxin (10−8 mol/L). Ach (10−5 mol/L) relaxation of Phe contraction was performed to confirm the presence of intact endothelium. In some experiments, the endothelium was mechanically removed by gently scraping the intimal surface of the IVC segment around the tip of forceps, and removal of the endothelium was confirmed by the absence of Ach relaxation.

Nitrite/Nitrate measurements

Endothelium-intact IVC segments were placed in 1.5 ml Krebs aerated with 95% O2 5% CO2 at 37°C and the solution was changed every 30 min for 1 hr. Samples for basal accumulation of nitrite (NO2−) formed from released NO were taken. IVC segments were treated with Ach or MMP-2 for 30 min, then rapidly removed, dabbed dry with filter paper and weighed. The incubation solutions were assayed for the stable end product of NO, NO2−. Briefly, samples of incubation solution (50 μl, in triplicate) were mixed in 96-well microplate with 100 μl Griess reagent. The chromophore generated from the reaction with NO2− was detected spectrophotometrically (535 nm) using SpectraMAX microplate reader (Molecular Devices, Sunnyvale CA). The concentration of NO2− was calculated using a calibration curve with known concentrations of NaNO2.

NO production

NO measurements were confirmed with 4-amino-5-methylamino-2′,7′-difluorescein (DAF-FM), an NO-sensitive fluorescent dye.18 IVC rings were placed in test tubes containing 2 mL Krebs and 7 μmol/L DAF for 45 min. Samples for basal accumulation of NO were taken. The IVC rings were treated with Ach or MMP-2 for 30 min then removed, dabbed dry with filter paper and weighed. The fluorescence of the incubation solutions was measured (50 μL, in triplicate) in a 96-well microplate using SpectraMAX microplate reader with excitation wavelength on 495 nm and emission wavelength of 520 nm.

Statistical Analysis

The data were analyzed and presented as mean±SEM. Data were analyzed using ANOVA followed by Scheffe's F test for comparison of multiple means. Student's t-test for unpaired and paired data was used for comparison of two means. Differences was considered statistically significant if P < 0.05.

RESULTS

MMP-2 induces relaxation of Phe contraction

In endothelium-intact IVC, Phe caused significant contraction that ranged between 0.5 to 0.6 g. Phe contraction showed a gradual decline with time and reached 82.0±2.4% of the initial control contraction in 30 min. MMP-2 (1 μg/ml) caused relaxation of Phe contraction to between 0.2 and 0.3 g or 39.5±10.5% of the control contraction in 30 min (n=5, P = 0.029) (Figure 1). To determine if MMP-2 acts via an endothelium-dependent mechanism, the effects of MMP-2 were compared in endothelium-denuded and intact IVC. Removal of the endothelium did not inhibit MMP-2 induced relaxation. Instead, MMP-2 caused greater inhibition of Phe contraction to 7.6±2.0% in endothelium-denuded (n=4, p=0.032) compared with intact IVC (Figure 1).

Figure 1.

Figure 1

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MMP-2 induced relaxation of phenylephrine (Phe) contraction in endothelium-intact IVC segments nontreated (A) or pretreated with L-NAME (10−4 mol/L) (B) or L-NAME+indomethacin (10−5 mol/L) (C) or endothelium-denuded IVC segments (D). When Phe (10−5 mol/L) reached a steady-state, MMP-2 (1 μg/ml) was added and its effect on Phe contraction was observed. The traces are representative of 3 to 5 experiments.

Effects of L-NAME on MMP-2 Induced IVC Relaxation

To determine if MMP-2 induced IVC relaxation involves the NO pathway, NO synthesis was blocked by the NOS inhibitor L-NAME (10−4 mol/l). In L-NAME pretreated IVC MMP-2 still caused significant relaxation of Phe contraction to 4.4±1.6% of control (n=3, P = 0.028). The MMP-2 induced relaxation of Phe contraction was significantly greater in the presence of L-NAME compared with that in the absence of L-NAME (p=0.046) (Figures 1 and 2).

Figure 2.

Figure 2

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Aggregate data evaluating the effect of MMP-2 on Phe contraction in endothelium-intact IVC segments nontreated or treated with L-NAME and indomethacin and in endothelium-denuded segments (-endo). Data represent mean±SEM of measurements in 3 to 5 experiments. * p<0.05 compared to control experiments in endothelium-intact tissues, and experiments in endothelium-blocked tissues.

Effect of MMP-2 on NO production

NO was assayed in IVC at basal levels and in the presence of MMP-2 (1 μg/ml) or Ach (10−5 mol/L). Ach caused significant increase in NO production above basal levels. MMP-2 did not significantly increase NO production, and appeared to cause a reduction in NO production compared to basal levels (n=3) (Figure 3).

Figure 3.

Figure 3

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Effect of MMP-2 on nitrite/nitrate production as measured by Griess reagent (A) and on NO production as measured by DAF-FM fluorescence (B). Basal, MMP-2 and Ach-induced NO production were measured. Data represent mean±SEM of measurements in 3 experiments.

Effect of indomethacin on MMP-2 induced IVC relaxation

To determine whether MMP-2 induced IVC relaxation involves prostacyclin (PGI2)-dependent relaxation pathway the effects of indomethacin, inhibitor of cyclooxygenases, were tested. In IVC pretreated with indomethacin (10−5 M) MMP-2 caused relaxation of Phe contraction to 49.8±3.5%, which was not different from that in tissues treated with MMP-2 alone (p=0.43). In IVC pretreated with indomethacin plus L-NAME, MMP-2 caused significant relaxation of Phe contraction to 6.5±1.2% of control (p<0.0001) (Figures 1 and 2).

Effect of 96 mmol/L KCl on MMP-2 induced IVC relaxation

To determine whether MMP-2 induced IVC relaxation involves hyperpolarization of vascular cells, we measured the effect of MMP-2 in the presence of high KCl depolarizing solution. MMP-2 did not cause any significant relaxation of KCL contraction (95.3±2.8%) (Figure 4). In contrast, addition of Ach caused significant relaxation of the KCl contraction.

Figure 4.

Figure 4

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Effects of MMP-2 during membrane depolarization by KCl, a condition that does not favor K+ efflux and cell hyperpolarization. High KCl (96 mmol/L) contraction was elicited, MMP-2 (1 μg/ml) was added and its lack of effect was observed. Data represent mean±SEM.

Effect of cromakalim (KATP activator)

To test whether MMP-2 induced relaxation involves hyperpolarization and activation of KATP channel, we examined if prior activation of KATP with cromakalim would prevent MMP-2 induced IVC relaxation. Addition of cromakalim (10−7 mol/L) caused significant relaxation of Phe contraction. Addition of MMP-2 caused further relaxation of IVC to the baseline, indicating that MMP-2 induced hyperpolarization and relaxation involves mechanisms other than the KATP channel (Figure 5).

Figure 5.

Figure 5

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Effect of cromakalim on MMP-2 induced IVC relaxation. IVC contraction to Phe was elicited. Control tissue (upper panel) was treated with vehicle Krebs. In other tissues (lower panel), cromakalim (10−7 mol/L) was added and IVC relaxation was observed. MMP-2 was then added and further IVC relaxation to baseline could be observed.

Effect of iberiotoxin (blocker of BKCa)

To further evaluate the role of K+ channels in MMP-2 induced IVC relaxation, the effect of blocking BKCa was tested. The addition of iberiotoxin (10−8 mol/L) to MMP-2 treated IVC caused significant attenuation of relaxation (12.1±3.6%) compared to the control MMP-2 induced relaxation (60.5±10.5%, P=0.0058, n=5) (Figure 6).

Figure 6.

Figure 6

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Effect of iberiotoxin on MMP-2 induced IVC relaxation. IVC contraction to Phe was elicited. Control tissue (top panel) was treated with vehicle Krebs. In other tissues (middle panel), addition of iberiotoxin (BKCa blocker) prevented MMP-2 induced relaxation of Phe contracted IVC. Cumulative effects of MMP-2 in the absence or presence of iberiotoxin are presented in bottom panel. *p<0.05

DISCUSSION

The present study has demonstrated that MMP-2 induces significant relaxation of IVC segments precontracted with Phe. Although the effects of MMPs on the vascular extracellular matrix are well-documented, little is known regarding the effects of MMPs on the endothelium and smooth muscle. Immunohistochemical studies have shown that MMPs are localized not only in the vein adventitia and extracellular matrix, but also in the endothelium and smooth muscle cells.14 The observed relatively rapid venous relaxation effects of MMPs suggest possible activation of endothelium-dependent relaxation pathway, or endothelium-independent inhibition of venous smooth muscle contraction, or both.

The enhanced MMP-induced relaxation in endothelium-denuded compared with intact veins suggests that an endothelium-derived contracting or relaxation factor(s) may interfere with MMP-2 induced vasodilation. Minimizing these endothelial factors (by mechanical removal of the endothelium or chemical blocking of endothelium-derived mediators) would lead to enhanced MMP-2 vasodilation. Studies have suggested that MMP-2 could cleave big endothelin-1 yielding a novel vasoconstrictor and thereby enhance vascular contraction, an effect that will not be manifested in endothelium-denuded tissues.19 MMP-2 induced venous relaxation could also be due to enhanced release of nitric oxide (NO), prostacyclin (PGI2) or endothelium-derived hyperpolarizing factor (EDHF).20

Studies have suggested a role of MMPs in blood flow-induced arterial dilation via interaction with NO, raising the possibility that MMPs may be coupled to eNOS stimulation and activation of the NO-cGMP venous relaxation pathway.21 We found that the NOS inhibitor L-NAME did not attenuate, and instead, enhanced MMP-2 induced IVC relaxation. Also, MMP-2 did not increase NO production as measured by Griess reagent and DAF fluorescence. Although the data suggest that MMP-2 induced venous relaxation may not involve increased NO production, they do not rule out possible interaction between the NO pathway and MMP. NO, being a major vasodilator, may downregulate the effects of MMP-2 on other endothelium-derived vasodilators, and NOS inhibition may unmask these effects. Also, studies have shown that NO inhibition may increase MMP-9 expression in rat vascular smooth muscle cells.22 MMP-2 may also directly, or indirectly through increased endothelin-1 production, induce NOS uncoupling and lead to increased superoxide production and decreased NO bioactivity.23,24 We have also found that the MMP-2 induced venous relaxation was not affected by the cyclooxygenase inhibitor indomethacin, suggesting that it does not involve increased PGI2 synthesis and activation of PGI2-cAMP relaxation pathway.

The inability of NOS and cyclooxygenase inhibition to block the MMP-2 induced venous relaxation raises the possibility of MMP-2 mediated release of EDHF leading to enhanced K+ efflux via K+ channels and venous smooth muscle hyperpolarization and relaxation. To test the possible role of EDHF, we examined whether MMP-2 induced venous relaxation is affected by changing the extracellular K+ concentration gradient or by K+ channel modulators. High extracellular KCl creates a K+ concentration gradient that does not favor K+ efflux through plasma membrane K+ channels. In the present study, MMP-2 did not inhibit venous contraction during membrane depolarization by high KCl, suggesting that MMP-2 induced relaxation likely involves hyperpolarization and activation of a K+ channel.

K+ channels include the ATP-sensitive K+ channel (KATP), large conductance Ca2+-activated K+ channel (BKCa), intermediate and small conductance Ca2+-activated K+ channel, voltage-gated K+ channels, and inward rectifier K+ channels.25 In human saphenous vein the K+ channel opener cromakalim works mainly by activating the KATP channel.26,27 If MMP-2 causes venous relaxation by activating the KATP channel, one would predict that following the addition of cromakalim MMP-2 should not cause any further IVC relaxation. MMP-2 caused further IVC relaxation in cromakalim-treated IVC, suggesting that MMP-2 induced venous relaxation involves K+ channels other than the KATP channel.

To test whether MMP-2 induced venous relaxation involves activation of BKCa we examined whether the effects of MMP-2 are reversed by iberiotoxin, a specific blocker of BKCa.25 Iberiotoxin at 10−8 M, which is 10-fold lower than its IC50 (10−7 M), essentially abolished MMP-2 induced IVC relaxation, supporting that MMP-2 induced IVC dilation is, in part, mediated through hyperpolarization and activation of BKCa. However, the data do not exclude possible involvement of other types of K+ channels in IVC relaxation.

The question arises as of how MMP-2 induced activation of K+ channels could cause venous relaxation. Activation of K+ channels likely causes smooth muscle hyperpolarization, and leads to decreased Ca2+ influx through voltage-gated channels. This is supported by a previous report which demonstrated that MMP-2 and MMP-9 cause aortic dilatation by inhibiting Ca2+ entry into aortic smooth muscle.16 Another question is how MMP-2 causes activation of K+ channels. Although MMP-2 does not appear to increase endothelium-derived NO or PGI2, it could still interact with the endothelium and induce the release of EDHF. MMPs have been shown to promote bradykinin release in the vein wall, which could in turn increase the release of endothelium-derived relaxing factors.28-30 Although the exact nature of EDHF is unclear, studies suggest that EDHF mediated responses could involve epoxyeicosatrienoic acids, which are epoxides of arachidonic acid generated by cytochrome P450 epoxygenases.31 Other possible EDHFs include hydrogen peroxide (H2O2).32 Whether inhibition of cytochrome P450 epoxygenases or H2O2 production prevents MMP-induced dilation would determine the importance of these EDHFs in the MMP-mediated effects.

MMP-2 could also directly activate the K+ channels. MMP may facilitate a conformational change in the BKCa channel from a closed state to an open state, during changes in voltage and intracellular Ca2+.33,34 Other possibilities include increases in Arg-Gly-Asp (RGD) tripeptide containing sequence as a result of collagen degradation by the protease activity of MMP-2. Soluble RGD peptide can then bind to smooth muscle αvβ3 integrin receptors and lead to activation of K+ channels, venous hyperpolarization and relaxation.35,36 This is supported by reports that the RGD peptide integrin signaling and vasodilatation may involve modulation of the voltage-gated and the inward rectifying K+ channels.36 MMP-2 may also activate K+ channels through specific protease-activated receptors (PARs). PARs are activated by serine proteases such as thrombin, trypsin, and tryptase.37,38 Trypsin causes an L-NAME/indomethacin resistant relaxation in mesenteric arteries of PAR2(+/+), but not PAR2(−/−) mice.39 The trypsin-induced PAR2-mediated relaxation was inhibited by KCl-precontraction, or pretreatment with apamin or charybdotoxin, blockers of small and intermediate Ca2+-activated K+ channels, respectively. These data suggest that PAR2-mediated relaxation of the mouse vasculature involves hyperpolarization of vascular smooth muscle and activation of Ca2+-activated K+ channels.39 Our recent studies have shown that TIMP-1 prevented MMP-2 induced relaxation of Phe contraction in rat, possibly due to inhibition of MMP-2 proteinase activity or TIMP-1 binding to integrins.40 Whether MMP-2 induced venous relaxation and activation of K+ channels involves RGD peptide-integrin signaling or PARs activation remains to be investigated.

It would be of interest to study the direct effects of MMP-2 on membrane potential and ion channel activities in isolated vascular smooth muscle cells. MMPs are matrix metalloproteinases that regulate the extracellular matrix (ECM) turnover. Isolated vascular smooth muscle cells lack a native ECM. An effect of MMPs through integrins (which are components of the ECM) would be minimized in the isolated vascular smooth muscle cell system. Also, isolating vascular cells often requires treating the blood vessels with collagenase/elastase. We have previously shown that elastase causes vascular smooth muscle relaxation, which could mask the effects of MMPs.41 Future studies would examine experimental protocols to isolate vascular smooth muscle cells using small concentrations of proteases so that the effects of MMPs are not compromised.

The present study focused on testing the effects of MMP-2 on venous function. Large amounts of MMP-2 have been detected in the plasma and venous tissues of patients with varicose veins. However, MMP-1, -3, -9, and -13 are also expressed in human varicose veins.13-15 Also, it is possible that some of the effects of MMP-2 could be due to activation of other endogenous MMPs.5 Future studies should examine whether the effects of MMPs on the mechanisms of venous relaxation are unique to MMP-2, or are caused by other MMPs. Also, whether the observed effects of MMP on IVC could also be demonstrated in other venous tissues such as the rat femoral vein and human saphenous vein should be examined in future experiments.

In conclusion, MMP-2 causes relaxation of Phe contraction in IVC segments by a mechanism involving hyperpolarization and activation of BKCa. A protracted MMP-2 induced venous relaxation could lead to progressive venous dilatation, varicose vein formation and chronic venous insufficiency.

ACKNOWLEDGEMENTS

This work was supported by grants from National Heart, Lung, and Blood Institute (HL-65998, and HL-70659).

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