Morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants

Abstract

Objectives

To investigate morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants sodium tetradecyl sulfate and polidocanol.

Methods

Samples of whole blood, isolated leukocytes, platelets, endothelial cells, and fibroblasts were incubated with varying concentrations of sclerosants. Whole blood smears were stained with Giemsa and examined by light and bright field microscopy. Phalloidin and Hoechst stains were used to analyze cytoplasmic and nuclear morphology by fluorescence microscopy. Endothelial cell and fibroblasts were analyzed by live cell imaging.

Results

Higher concentrations of sclerosants induced cell lysis. Morphological changes in intact cells were observed at sublytic concentrations of detergents. Low concentration sodium tetradecyl sulfate induced erythrocyte acanthocytosis and macrocytosis, while polidocanol induced Rouleaux formation and increased the population of target cells and stomatocytes. Leukocytes showed swelling, blebbing, vacuolation, and nuclear degradation following exposure to sodium tetradecyl sulfate, while polidocanol induced pseudopodia formation, chromatin condensation, and fragmentation. Platelets exhibited pseudopodia with sodium tetradecyl sulfate and a “fried egg” appearance with polidocanol. Exposure to sodium tetradecyl sulfate resulted in size shrinkage in both endothelial cell and fibroblasts, while endothelial cell developed distinct spindle morphology. Polidocanol induced cytoplasmic microfilament bundles in both endothelial cell and fibroblasts. Patchy chromatin condensation was observed following exposure of fibroblasts to either agent.

Conclusion

Detergent sclerosants are biologically active at sublytic concentrations. The observed morphological changes are consistent with cell activation, apoptosis, and oncosis. The cellular response is concentration dependent, cell-specific, and sclerosant specific.

Keywords

Sclerotherapy sclerosants endothelial cells blood cells

Introduction

Sodium tetradecyl sulfate (STS) and polidocanol (POL) are detergent sclerosants administered at a range of concentrations to treat a variety of vascular conditions such as incompetent veins, vascular malformations, and oesophageal varices. The aim of sclerotherapy is to induce endothelial cell (EC) lysis resulting in vessel occlusion with the ultimate aim of inducing endovascular fibrosis.

The lytic effect of sclerosants is not limited to EC. Our group demonstrated lysis of erythrocytes, leukocytes, and platelets at concentrations specific to cell types and dependent on the diluting media.^1,2 Plasma proteins and membrane lipids deactivate the sclerosants resulting in a massive drop in the active concentration and a reduced lytic effect on ECs.² Sclerosant-induced lysis occurs at a minimum lytic concentration.^1,3 The biological effect of detergent sclerosants on living cells is not limited to lysis and is more interesting at sublytic concentrations. For example, we demonstrated platelet activation and release of platelet microparticles at low concentrations of both sclerosants.^4,5 More recently, we demonstrated release of pro-inflammatory and proangiogenic cytokines at similarly low sublytic concentrations of sclerosants, indicative of leukocyte activation.⁶

Given these findings, we hypothesized that detergent sclerosants can cross the lipid membranes to induce cytoplasmic and nuclear changes in exposed cells at sublytic concentrations. In the present study, we looked for microscopic evidence of possible effects on cell organelles and nuclei. These investigations focused on cells that may be directly or indirectly exposed to detergent sclerosants, i.e. vascular (ECs and fibroblasts) and circulating blood cells (erythrocytes, leukocytes, and platelets).

Materials and methods

Materials

The following were used in this study: STS was obtained as FIBRO-VEIN 3% (w/v), 47.4 mM (Australian Medical and Scientific, NSW, Australia), POL as AETHOXYSKLEROL 3% (w/v), 51.5 mM (Kreussler, Wiesbaden, Germany), human umbilical vein ECs, pooled (HUVECp), and adult human dermal fibroblasts (HDFa) (Life Technologies, CA, USA) were obtained as pooled samples in 10% dimethyl sulfoxide medium. Paraformaldehyde (PFA) and hexamethyldisilazane (HMDS) (ProSciTech, QLD, Australia), bovine serum albumin (BSA), and poly-l-lysine solution 0.1% (w/v) (Sigma Aldrich, St. Louis, USA). Fluorescent microscopy antibodies: Alexa Fluor 647 Phalloidin, Hoechst, (Invitrogen, CA, USA). Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Triton-x-100 (Becton-Dickinson, NJ, USA).

Sample collection for light and fluorescence microscopy

Whole blood (WB) was collected by normal standard procedures following informed consent from healthy volunteers taking no medications or supplements. These samples were collected in 10 ml EDTA tubes (BD Vacutainer, K2E 18.0 mg) to prepare WB smears stained with Giemsa dye and to isolate leukocytes and in 3 ml sodium citrate tubes (BD Vacutainer, 0.105 M) to isolate platelets.

Sample collection for scanning electron microscopy

Samples of post-sclerotherapy coagulum (sclero-coagulum; “trapped blood”) were collected from patients who had undergone sclerotherapy with STS 1.5%, six to eight weeks prior to collection. Drainage of the sclero-coagulum was performed as part of the routine clinical management of patients and samples were collected for analysis following an informed consent. Briefly, treated varicose veins were examined on ultrasound and segments containing sclerocoagulum were identified. Following subcutaneous administration of a local anaesthetic (5–10 ml, xylocaine 1% plus adrenalin 1:100,000), the sclero-coagulum was collected in a 20 ml plastic syringe via a 19 g needle and immediately processed for electron microscopy.

Sample preparation

A. Leukocyte isolation

Two milliliters of red blood cell lysis buffer (0.155 M NH₄Cl, 0.009 M KHCO₃, and 1.8 ml EDTA disodium salt) was added per 100 µl of WB in 15 ml centrifuge tubes, incubated at room temperature, and protected from light for 10 min. Samples were then centrifuged at 400 g for 10 min. The supernatant was discarded and the red cell lysis step was repeated. Cell concentration was adjusted to 1 × 10⁶ cells/ml using a haemocytometer.

B. Platelet isolation

Platelets were obtained as described previously.¹ Briefly, platelet-rich plasma was generated by centrifugation of WB for 10 min at 150 g. Acidifying buffer and apyrase were added and cells were incubated for 5 min. Cells were then centrifuged at 1000 g for 10 min and resuspended in 5% BSA.

C. EC incubation

HUVECs were incubated in HUVEC culture medium containing M199 (Invitrogen, CA, USA), 20% foetal bovine serum (FBS) (SAFC Biosciences, KS, USA), endothelial cell growth factor 30 µg/ml (Sigma–Aldrich, MD, USA), heparin 100 µg/ml (Pharmacia and Upjohn, USA), l-glutamine 10 mM (Invitrogen, CA, USA), penicillin, and streptomycin 10 µg/ml (Invitrogen, CA, USA), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 25 µg/ml (Invitrogen, CA, USA) at a concentration of 1.25 × 10⁴ viable cells/ml in flasks. Medium was changed every 48 h.

D. Fibroblast incubation

HDFa cells were incubated in supplemented medium (10% FBS, 100 U/ml penicillin/streptomycin, 2 mM l-glutamine, in Dulbecco's Modified Eagle Medium - low glucose [DMEM-LG]) at 37℃, 5% CO₂ in a cell culture incubator. The culture medium was changed every 48 h and the subculturing of cells occurred once confluence was achieved.

Incubation with sclerosants and microscopic analysis

A. WB cell morphology

Erythrocyte morphology was assessed following sclerosants incubation at increasing concentrations (0.075%, 0.15%, 0.30%, 0.60%, and 1.2%) with WB for 5 min. A blood smear was generated and stained using Giemsa dye ensuring the films had three zones: the body (near the point of blood application), the monolayer (zone between the body and the feathered edge), and the feathered edge (the most distant area from the point of application). Blood cell morphology was assessed using light microscopy (ColourVueIII, Olympus) and bright field microscopy (Leica DM 5500). Köhler illumination was adjusted evenly for all samples. Samples were then visualized with an Olympus LCA chN 40 × /0.55PhP/∞/1/FN22 and images were taken from the monolayer area to ensure consistent quantification of the morphological changes. Cell quantification was done manually in different fields.

B. Scanning electron microscopy analysis

Samples of sclerocoagulum were rinsed three times with cacodylate buffer 0.05 M and fixed with 2% glutaraldehyde in cacodylate buffer overnight. Samples were rinsed again with cacodylate buffer three times before they underwent a dehydration process. Samples were then submerged into different concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 95%) for 10 min and three more times with 100% ethanol for 20 min to dehydrate them. Under the fume hood, samples were then put into 50% HMDS (500 µl absolute ethanol and 500 µl HMDS) for 10 min and three more times in 100% HMDS for 15 min. HMDS was allowed to evaporate overnight at open air. Samples were then mounted and coated with gold and analyzed in a scanning electron microscope (Hitachi SEM 3400X).

Isolated leukocytes were fixed with 2% glutaraldehyde in cacodylate buffer at room temperature for 2 h and washed three times with cacodylate buffer. Cells were gradually dehydrated using series of ethanol (30–100%). Samples were then dried with HMDS for 2 min and culture dishes were placed in a desiccator to air dry. Coverslips were then mounted carefully onto an aluminum pin stub using carbon tape and coated with 15-nm layer of gold at 2 mA for 2 min. Samples were analyzed in a scanning electron microscope (Hitachi SEM 3400X).

C. Leukocyte, platelet, EC, and fibroblast morphology

Cells were resuspended in 5% BSA and incubated in culture plates containing poly-l-lysine precoated coverslips for 30 min to allow cells to adhere to the coverslips. Coverslips were washed once with PBS to remove unattached cells and subsequent treatments were administered (STS or POL) for 5 min. Two percent PFA/ Phosphate-buffered saline (PBS) was added for 10 min at room temperature to fix the cells and then blocked with 3% BSA/PBS for 30 min. Cells were incubated for 45 min with Alexa Fluor 647 Phalloidin to stain the cytoskeleton and Hoechst to stain chromatin. Immunostained cells were rinsed and coverslips were mounted using VectaShield mounting media and sealed with nail polish. The slides were visualized using a Leica DM 5500 (IF) fluorescent microscope. Each channel was recorded independently and pseudocolor images were generated and superimposed. Final images were processed and analyzed by the Image J software (National Institutes of Health, NIH).

D. Live cell imaging analysis

Cells resuspended in 5% BSA were placed on 12-well plates precoated with poly-l-lysine and placed into an incubator for 20 min to allow cells to adhere. Samples were then placed into a live cell imaging microscope (Leica DMI 6000) preset at 37℃ with 5% CO₂. Samples were stimulated with increasing concentrations (0.075%, 0.15%, 0.30%, 0.60%, and 1.2%) of either STS or POL. Images were obtained every 15 min over a period of 3 h. Results were analyzed by the Image J software v1.46 (National Institutes of Health, NIH, USA).

Results

A. Erythrocytes

Erythrocyte lysis was evident at STS concentrations≥0.6% and POL≥1.2%. At lower concentrations, both agents induced morphological changes in erythrocytes (Figure 1(a)). STS-induced erythrocyte acanthocytosis (18% of total red cell population) and macrocytosis (6–16% of total population) (Figure 1(b) I–II). POL induced a rise in the population of target cells (16%) and stomatocytes (20%) at 0.075% and 0.3%, respectively (Figure 1(b) III–IV). Erythrocyte aggregation was observed at 0.075% POL culminating in Rouleaux formation at ≥0.15% (Figure 1(b) V).

Figure 1.

Effect of detergent sclerosants in WB. (a) Samples of WB were incubated with varying concentrations of STS or POL for 5 min before staining with Giemsa stain. (a) Cartoon representation of typical cells stimulated with STS and POL at sublytic concentrations. (b) I. Acantocytosis (thin arrows), II. Macrocytosis (arrow heads) appearing at STS concentrations of 0.075% and 0.15–0.30%, respectively. Images III–V represent target cells (thick arrow), stomatocytes (thick head arrow), and Rouleaux formation induced by POL at 0.075%, 0.15%, and 0.30%, respectively. (c) Bright-field microscopy images of representative cells for each group. STS-induced blunt cytoplasmic prolongations (spiculations) characteristic of acanthocytes. POL-induced stomatocyte formation. (d) Scanning electron micrographs of sclero-coagulum (“trapped blood”) in patients treated with 1.5% STS six to eight weeks before. Lower magnification image (left) shows an increased number of acanthocytes (arrows). Higher magnification image (right) shows an acanthocyte trapped in fibrin strands.

B. Leucocytes

Leukocyte lysis was evident at STS and POL concentrations ≥0.3%. At lower concentrations (0.075% and 0.15%), both agents induced dramatic morphological changes in isolated leukocytes (Figure 2). STS-induced swelling followed by formation of large and in some occasions multiple blebs devoid of organelles with no obvious cytoplasmic disintegration (Figure 2(b,c)). The nuclei underwent swelling followed by degradation (Figure 2(d)). WB smears showed leukocyte vacuolation (Figure 2(e)). POL induced cell shrinkage with cytoplasmic budding, which evolved to tail-like elongation (Figure 2a,b). The nuclei exhibited patchy ring-like chromatin condensation at early stages followed by chromatin fragmentation (Figure 2(d)).

Figure 2.

Fluorescence microscopy of representative leukocytes following exposure to detergent sclerosants. (a) Cartoon representation of typical cells stimulated with STS and POL at sublytic concentrations. (b) Cytoplasmic morphology. Leukocytes stimulated with STS at 0.15% showed single or multiple cytoplasmic blebs. Cells stimulated with POL at 0.15% exhibited tail-like elongation. Cells were stained with Alexa Fluor 647 Phalloidin (red) and Hoechst (blue). (c) Scanning electron micrographs of isolated leukocytes stimulated with STS and POL at 0.075%. (d) Nuclear morphology. Cells stimulated with STS at 0.15% show DNA degradation, whereas cells stimulated with POL 0.15% show a ring-like chromatin condensation and chromatin fragmentation. (e) Light microscopy micrograph of WB sample treated with STS at 0.15%. Arrow shows a leukocyte with vacuolation. Samples were stained with Giemsa.

C. Platelets

Platelet lysis was evident at STS concentrations ≥ 0.6% and POL ≥ 1.2%. At 0.075%, STS induced pseudopodia formation (Figure 3(b–d)) and at 0.15%, cells were rounded and showed swelling. Platelets stimulated with POL at 0.075% showed swelling and at 0.15% developed a “fried egg” appearance with rounded protrusions (Figure 3(b,c)).

Figure 3.

Morphological changes in platelets following exposure to detergent sclerosants. (a) Cartoon representation of typical cells stimulated with STS and POL at sublytic concentrations. (b) Fluorescence micrographs of platelets stimulated with STS (0.075%) showed multiple fine and elongated spiculations. Platelets stimulated with POL (0.15%) were spread and showed elongation. (c) High magnification images of representative cells for each group. Cells were stained with Alexa Fluor 647 Phalloidin. (d) Scanning electron micrograph of sclero-coagulum from patients treated with STS showing two platelets with pseudopodia.

D. ECs

Endothelial lysis was observed at STS concentrations ≥ 0.6% and POL ≥ 0.3%. EC exposed to 0.075% STS shrunk in size, underwent a shape change from polygonal to spindle morphology, and formed cell clumps (Figure 4(b)). POL at 0.075% induced cytoplasmic microfilament bundles and cytoplasmic disruption (Figure 4(b)). Chromatin changes were not evident with either agent. In live cell imaging (Figure 4(c)), cells stimulated with STS at 0.075% developed pseudopodia (Supplementary video 1), at 0.15% showed multiple small blebs (Supplementary video 2), and at 0.30% multiple big cytoplasmic blebs (Supplementary video 3). Cells stimulated with low concentrations of POL (0.075%, 0.15%, and 0.30%) showed shrinkage (Figure 4(d)).

Figure 4.

Morphological changes in endothelial cells stimulated with detergent sclerosants. (a) Cartoon representation of typical cells stimulated with STS and POL at sublytic concentrations. (b) Fluorescence micrograph of cells stimulated with STS and POL at 0.075%. STS-induced spindle-like shape change, cell shrinkage, and cytoplasmic disruption. POL induced an increase in microfilament bundle appearance. Both STS and POL did not show significant nuclear changes in EC. (c) Live cell imaging sequences of EC treated with STS at I. 0.075%, showing filopodia formation and shrinkage of the cells. II. 0.15%, showing the formation of multiple dynamic and small blebs within neighbor cells, and III. 0.30%, showing the development of multiple static big blebs. Live cell imaging sequences of EC treated with POL at different concentrations I. 0.075%, II. 0.15%, and III. 0.30%. The three concentrations denote cell shrinkage. Images represent 15 min intervals.

E. Fibroblasts

Fibroblast lysis was observed at STS concentrations ≥ 0.6%. STS-induced pseudopodia formation at low concentrations (0.075–0.15%) and cell shrinkage at higher concentrations (0.30%). POL-induced pseudopodia formation at 0.075% and 0.15% and microfilament bundles at higher concentrations (0.30–1.2%) (Figure 5(b)). Cell lysis was not evident at any given concentration of POL. A chromatin ring surrounding the nucleus was observed at lower concentrations of both agents (Figure 5(c)). Patchy chromatin condensation was observed with both STS (0.075% and 0.3%) and POL (0.075%, 0.15%, and 0.3%).

Figure 5.

Morphological changes in fibroblasts stimulated with detergent sclerosants. (a) Cartoon representation of typical cells stimulated with STS and POL at sublytic concentrations. (b) Fluorescence micrograph showing cytoplasmic changes. STS-induced filopodia formation. Microfilament bundles were more evident with POL. (c) Nuclear changes. Both agents showed ring-like chromatin condensation. Cells were stained with Alexa Fluor 647 Phalloidin (red) and Hoechst (blue). (d) Live cell imaging sequences of fibroblasts treated with STS at I. 0.075%, showing the development of multiple static big blebs II. 0.15%, showing cell shrinkage on cluster of cells, and III. 0.60%, showing cell lysis, demonstrated by cells rupture. (e) Live cell imaging sequences of fibroblasts treated with POL at different concentrations I. 0.015%, showing bleb formation, II. 0.30% showing cell shrinkage, and III. 0.60% showing cell degranulation. Images represent 15 min intervals.

In live cell imaging, cells stimulated with STS at lower concentrations (0.075%, 0.15%, and 0.30%) showed swelling and bleb formation (Figure 5(d) and Supplementary video 4). At higher concentrations (0.60% and 1.2%), only cell debris was present. Cells stimulated with POL at 0.075% and 0.15% showed swelling and bleb formation (Supplementary video 5). Cells stimulated with POL at 0.30%, 0.60%, and 1.2% showed shrinkage. Degranulation of fibroblasts was observed at 0.60% of POL (Supplementary video 6).

Discussion

Cells undergo shape change in response to a range of physical, chemical, and biological stimuli.⁷ In this study, we observed a range of nuclear and cytoplasmic changes in vascular and circulating blood cells exposed to sublytic concentrations of detergent sclerosants. In general, a pattern emerged where cell lysis occurred at concentrations higher than a minimum (lytic) concentration of detergents. Morphological evidence of other forms of cell death (oncosis, apoptosis, or senescence) was observed at sublytic concentrations and morphological features of cellular activation were detected at even lower (sublethal) concentrations. Morphological changes detected in this study were concentration dependent, cell specific, and sclerosant specific. The observed progression of activity is consistent with the gradual effect of detergents on lipid membranes where the final outcome is determined by the detergent to membrane lipid ratio.³

Erythrocytes comprise 95% of all circulating blood cells. As expected, we detected erythrocyte lysis at higher concentrations of both sclerosants (≥0.6% STS, ≥1.2% POL). Erythrocytes exposed to lower concentrations of detergents underwent morphological changes indicative of alterations in membrane composition, membrane expansion, and increased cell permeability. A common finding at these lower concentrations was erythrocyte acanthocytosis. These cells are spherocytic erythrocytes characterized by spike-like surface protrusions. They are less flexible and have a reduced surface area for the exchange of oxygen. Following exposure to sublytic concentrations of STS (but not POL), acanthocytes formed almost 20% of all erythrocytes. In addition, acanthocytes were observed trapped within strands of fibrin in ex vivo samples of sclero-coagulum collected from veins treated with STS. STS-induced acanthocytosis may be due to a number of reasons. Assimilation of excess lipids (cholesterol or sphingomyelin) in the outer half of the erythrocyte membrane is often the cause of acanthocyte morphology and lipid metabolic disorders arising from apolipoprotein-B (Apo B) deficiency result in acanthocytosis. We have previously shown STS (and not POL) to induce a precipitate of Apo B⁴ and fibrinogen which would result in a localized deficiency of Apo B. In addition, acanthocytosis has been attributed to variants in the anion transporter B and 3 protein.⁸ STS, being an anionic molecule possibly interferes with the function of this anion transporter. Finally, physical insertion of detergent monomers results in expansion of the exoplasmic aspect of the lipid membranes, previously associated with the spiked shape of acanthocytes.⁹

In addition, low concentration STS induced macrocytosis. Macrocytes are uniformly round and enlarged erythrocytes with a diameter greater than 9 µm. Macrocytosis is associated with increased membrane permeability and excess intracellular fluid and is found in conditions such as alcoholism.¹⁰ In this study, the increase in erythrocyte size was possibly caused by progressive and gradual incorporation of STS monomers in the lipid membrane.³ This would result in increased permeability and increased intracellular fluid. Macrocytosis was seen in 6–16% of all erythrocytes and compared to acanthocytosis was a less common feature.

Low-concentration POL increased the population of target cells and stomatocytes. Target cells are erythrocytes with the appearance of a bull’s eye (or target) and represent an increased surface to volume ratio. Target cells are found in conditions such as haemoglobinopathies and thalassemias. Stomatocytes are erythrocytes demonstrating an elongated area of central pallor appearing as a “stoma” which indicates a reduction of the surface area to cell volume ratio.¹¹ Stomatocytes are reported in conditions such as haemolytic anaemia and acute alcoholism. The underlying mechanism responsible for these changes is unclear, but may due to incorporation of POL monomers in the lipid membrane. At lower detergent to lipid concentration ratios, incorporation of the detergent would result in an increase in surface area but at higher concentrations, the disruption of membrane lipid would result in increased membrane permeability, cytoplasmic swelling, and a rise in cell volume. Other nonionic detergents such as Triton X-100 interact in the same way with erythrocyte membranes.¹²

Further to the above, low-concentration POL induced erythrocyte aggregation in Rouleaux. Rouleaux are stacks of erythrocytes in long chains formed under low shear or static flow conditions. Rouleaux formation is encouraged by nonionic environments as well as the presence of fibrinogen and other acute phase proteins.¹³ In humans, as against some other species, erythrocytes have a negative surface charge due to ionized sialic acid located on exterior glycoproteins. Under normal circumstances, this negative charge generates a repulsive force between the cells. A decrease in sialic acid content of the erythrocyte membrane due to oxidative stress results in a reduction of the repulsive force and an increase in cell aggregation.¹⁴ Incorporation of POL, a nonionic surfactant in the cell membrane may reduce this electrorepulsive force, encouraging cell aggregation. As expected, the anionic detergent STS did not promote Rouleaux formation.

The clinical relevance of sclerosant-induced generation of deformed eythrocytes and Roulaux formation is unknown. Normal erythrocytes are highly flexible, biconcave cells designed to optimize laminar flow. Blood viscosity under laminar flow conditions is influenced strongly by erythrocyte deformability and Rouleaux formation. It can be speculated that the local cumulation of deformed erythrocytes in larger vessels would be of little significance. However in small cutaneous venules, cumulation of such cells and Rouleaux formation would result in microvascular hyperviscosity. Stagnant flow observed in post-sclerotherapy telangiecatic matting and cyanotic erythema may be a manifestation of the findings described here. Further haemorheological studies are required to explore the role of deformed and aggregated erythrocytes in the pathogenesis of post-sclerotherapy complications.

Leukocytes comprise only 0.1% of all circulating cells. As expected, leukocytes exposed to higher concentrations of sclerosants (≥0.3%) underwent lysis. At lower sublytic concentrations, both sclerosants appeared to induce other forms of leukocyte death (oncosis, apoptosis). Cells exposed to sublytic concentrations (0.075–0.15%) of STS showed membrane blebs, cytoplasmic swelling, nuclear swelling, and nuclear degradation (karyolysis). Although the morphological features of apoptosis and oncosis may overlap, the presence of cytoplasmic swelling and karyolysis is a typical feature of oncosis.^15–17 Oncosis is a process of passive cellular death due to impairment of the ionic pumps. Oncosis is distinct from simple lysis which is secondary to the breakdown of cell membranes and cellular disintegration. Other agents known to induce oncosis include the antimalarial drug artesunate and the cytotoxic agent kahalalide F.^16,18 STS, an anionic detergent, possibly interferes with the function of the ionic pumps resulting in oncosis.

Low-concentration POL (0.15%) on the other hand appeared to induce morphological changes suggestive of leukocyte apoptosis as evident by cell shrinkage, ring-like chromatin condensation, and chromatin fragmentation.^19,20 Other nonionic detergents such as η-octyl glucoside, Triton X-100, and Tween 20 have been extensively used to activate the intrinsic pathway of apoptosis in different type of cells such as thymocytes, splenocytes, and HeLa cells.²¹ This is mediated by activation of BAX, a pro-apoptotic member of the BCL-2 family of the intrinsic pathway of apoptosis. The molecular mechanism of BAX activation by detergents is unknown.

In addition to inducing nonlytic cellular death, both agents induced other morphological changes in exposed leukocytes. Low-concentration STS (0.15%) induced leukocyte vacuolation in WB sample. Vacuolation typically follows lysosomal degranulation, hence STS possibly disrupts the lysosomal lipid membranes resulting in degranulation and the subsequent vacuolation. This is further evidence of the intracytoplasmic activity of this detergent sclerosant. POL at lower concentrations of 0.075–0.15% induced leukocyte pseudopodia formation. These are projections of cellular membranes observed in activated leukocytes used for motility and migration.²² Leukocytes release inflammatory and angiogenic cytokines following exposure to low concentrations of detergent sclerosants.⁶ Pseudopodia formation is further evidence of leukocyte activation at lower sublethal concentrations of POL.

Platelets comprise 4.9% of all circulating cells. Platelet lysis was observed at ≥0.6% STS and 1.2% POL. At sublytic concentrations, STS induced pseudopodia formation, while POL induced cytoplasmic protrusions and “fried egg” morphology. These morphological features are indicative of platelet activation^23,24 and consistent with our previous reports of platelet activation at low concentrations of both sclerosants.⁵ Morphological features of other forms of cell death (e.g., apoptosis) were not evident in platelets.

In this study, high-concentration STS induced endothelial lysis, whereas features of endothelial oncosis were observed at sublytic concentrations and endothelial activation at even lower (sub-lethal) concentrations. Under static conditions, EC has a uniform polygonal shape. Lysis was observed at STS concentrations ≥ 0.6% and was demonstrated by the presence of cellular debris. At 0.30%, EC developed big static membrane blebs consistent with cellular oncosis. At slightly lower concentrations (0.15%), EC developed multiple dynamic small blebs. This feature is observed in the initial phase of EC spreading, mitosis, and cell migration.²³ When stimulated at sublytic concentrations of STS (0.075%), EC showed a reduction in cell size and developed spindle morphology, pseudopodia elongations, and cell clumping, features previously associated with EC activation.²⁴

POL induced EC lysis at 0.3–1.2%. At sublytic concentrations (0.075–0.15%), POL induced cell shrinkage and the microfilament bundles were more evident. Microfilament bundles are associated with cellular senescence, an irreversible growth arrest on cells that encounter oncogenic stress. Such cells lose their proliferative capacity despite continued viability and metabolic activity and may release cytokines and other mediators.²⁵ Cell senescence can be induced by radiation and chemotherapeutic drugs such as doxorubicin.²⁶

Fibroblasts were lysed at STS concentrations ≥ 0.6%. Similar to the effect on EC, fibroblasts stimulated at 0.3% showed cell shrinkage and chromatin condensation (features suggestive of apoptosis) followed by swelling and cytoplasmic blebbing at 0.15–0.3% (features suggestive of oncosis). Those exposed to lower STS concentrations (0.075–0.15%) showed pseudopodia formation indicative of cell-to-cell interaction and locomotion.

Fibroblasts were lysed at POL concentrations of 1.2%. At concentrations ranging from 0.3% to 1.2%, the microfilament bundles in the cytoplasmic membrane were more evident, similar to the ones present on EC and could be indicative of cellular senescence. Chromatin condensation and cell shrinkage were also evident suggestive of cellular apoptosis. At lower concentrations (0.075–0.15%), fibroblasts showed pseudopodia formation. On live cell imaging, fibroblasts stimulated with POL 0.6% showed features suggestive of fibroblast degranulation. We suggest that this finding could be related to cell apoptosis where apoptotic bodies are released, however more studies are needed to clarify our findings.

As evident from these findings, the morphological changes induced by STS were distinct from POL. These differences may be due to the ionic charge of the agents or the effect of excipients in the commercial solutions. For example, Aethoxysklerol (POL) contains ethyl alcohol as an excipient, whereas FIBRO-VEIN (STS) contains benzyl alcohol. Further research is required to determine the role of excipients in experimental observations reported by this group and others.

Clinically, low concentrations of sclerosants may be directly injected or may be achieved downstream from the initial point of intravascular administration of the higher concentrations of the agents.² Hence, possible biological effects may be detected downstream and distal from the initial point of entry. Activated or deformed cells may enter the systemic circulation, although at low numbers, and may be detected in blood films by hematopathologist who need to be aware of these effects.

This study had a number of limitations. These morphological studies confirm the biological activity of sclerosants at low sublytic concentrations. Other studies are underway by this group to confirm the present findings and to investigate the mechanism of the observed changes. The concentrations discussed here were not absolute as different populations of cells underwent different morphological changes in a simultaneous fashion.

In summary, detergent sclerosants induced a large number of morphological changes on vascular and circulating blood cells. Both agents induced cell-specific changes consistent with increased cell permeability, as expected of the interaction of sclerosants with cell membranes. The observed morphological changes are indicative of a number of possible processes including cell activation, oncosis, and apoptosis. Given these findings, low-concentration detergent sclerosants are capable of inducing cellular death by mechanisms other than cellular lysis. Further studies are underway to elucidate the mechanism of action of these agents in cells studied here.

Footnotes

Acknowledgements

We acknowledge Xiaotong (Cherryl) Tan, Kelvin Cheung, and Jessica Gerbelli for assisting with the cell cultures and David Du for pictorial presentations.

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: This research was funded by the Academy of Phlebology and supported by the Dermatology, Phlebology, and Fluid Mechanics Research Laboratory, St Vincent’s Centre for Applied Medical Research, Sydney, Australia, and by NIH grant HL090774.

References

Parsi

Exner

Connor

. The lytic effects of detergent sclerosants on erythrocytes, platelets, endothelial cells and microparticles are attenuated by albumin and other plasma components in vitro. Eur J Vasc Endovasc Surg 2008; 36: 216–223.

Connor

Cooley-Andrade

Goh

. Detergent sclerosants are deactivated and consumed by circulating blood cells. Eur J Vasc Endovasc Surg 2014. Epub. DOI: 10.1016/j.ejvs.2014.12.029.

Parsi

. Interaction of detergent sclerosants with cell membranes. Phlebology 2014; pii: 0268355514534648–0268355514534648.

Parsi

Exner

Connor

. In vitro effects of detergent sclerosants on coagulation, platelets and microparticles. Eur J Vasc Endovasc Surg 2007; 34: 731–740.

Parsi

Connor

Pilotelle

. Low concentration detergent sclerosants induce platelet activation but inhibit aggregation due to suppression of GPIIb/IIIa activation in vitro. Thromb Res 2012; 130: 472–478.

Cooley-Andrade

Jothidas

Goh

. Low concentration detergent sclerosants stimulate white blood cells and release pro-inflammatory and proangiogenic cytokines in vitro. Journal of Vascular Surgery 2014; 2: 433–440.

Choi

. Hyperthermia-induced pseudopodia (thermopodia) are observed in neutrophils from febrile patients. Int Hematol 2003; 78: 266–267.

Bruce

Tanner

. Structure-function relationship of band 3 variants. Cell Mol Biol 1996; 42: 953–973.

Siegl

Hamminger

Jank

. Alterations of red cell membrane properties in neuroacanthocytosis. PLOS 2013; 8: e76715.

10.

Ford

. Red blood cell morphology. Int Jnl Lab Hem 2013; 35: 351–357.

11.

Gallagher

Chang

Retting

. Altered erythrocyte endothelial adherence and membrane phospholipid asymmetry in hereditary hydrocytosis. Blood 2003; 101: 4625–4627.

12.

Prete

Domingues

Meirelles

. Multiple stages of detergent-erythrocyte membrane interaction—a spin label study. Biochimica et Biophysica Acta 2011; 1808: 164–170.

13.

Neu

Meiselman

. Depletion-mediated red blood cell aggregation in polymer solutions. Biophys J 2002; 83: 2482–2490.

14.

Mehdi

Singh

Rizvi

. Erythrocyte sialic acid content during aging in humans: correlation with markers of oxidative stress. Disease Markers 2012; 32: 179–186.

15.

Van Cruchten

Broeck Van Den

. Morphological and biochemical aspects of apoptosis, oncosis and necrosis. Anat Histol Embryol 2002; 31: 214–223.

16.

Hallock

Tang

Buja

. Aurintricarboxylic acid inhibits protein synthesis independent, sanguinarine-induced apoptosis and oncosis. Toxicol Pathol 2007; 35: 300–309.

17.

Pryde

Walkers

Rossi

. Temperature-dependent arrest of neutrophil apoptosis. J Biol Chem 2000; 275: 33574–33584.

18.

Suarez

Gonzalez

Cuadrado

. A new marine-derived compound, induces oncosis in human prostate and breast cancer cells. Mol Cancer Ther 2003; 2: 863–872.

19.

Tone

Sugimoto

Tanda

. Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis. Exp Cell Res 2007; 313: 3635–3644.

20.

Ivashyna

Garcia-Saez

Ries

. Detergent-activated Bax protein is a monomer. J Biol Chem 2009; 284: 23935–23946.

21.

Hsu

Youle

. Nonionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem 1997; 272: 13829–13834.

22.

Xue

Janzen

Knecht

. Contribution of filopodia to cell migration: a mechanical link between protrusion and contraction. Int J Cell Biol 2010. Epub. DOI: doi: 10.1155/2010/507821.

23.

Norman

Bruges

Sengupta

. Cell blebbing and membrane area homeostasis in spreading and retracting cells. Biophys J 2010; 99: 1726–1733.

24.

Malek

Izumo

. Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress. J Cell Sci 1996; 109: 713–726.

25.

Kuilman

Michaloglou

Mooi

. The essence of senescence. Genes Dev 2010; 24: 2463–2479.

26.

Roninson

. Tumor cell senescence in cancer treatment. Cancer Res 2003; 63: 2705–2715.

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