Journal of Human Hypertension (2010) 24, 684–686 & 2010 Macmillan Publishers Limited All rights reserved 0950-9240/10 www.nature.com/jhh RESEARCH LETTER Leukocyte membrane bleb and pseudopod formation in hypertension

Journal of Human Hypertension (2010) 24, 684–686; individuals with elevated BP, and that in response to doi:10.1038/jhh.2010.41; published online 22 April 2010 exercise challenge, pseudopod formation would be reduced in normal BP subjects but increased in subjects with elevated BP. Leukocyte activation, including adhesion molecule Twenty-two sedentary, healthy volunteers with expression, oxygen radical generation and, in normal or elevated BP (age 26–60 years, 9 female) animal studies, pseudopod formation, is a hallmark gave written informed consent. The protocol was of hypertension. This study examined pseudopod approved by the University of California, San Diego and bleb formation and demonstrates that leuko- Institutional Review Board. All subjects underwent cytes from hypertensive individuals are more a history and physical examination by a physician susceptible to produce membrane blebs than leuko- and a normal electrocardiogram was confirmed. cytes from normotensive individuals. Bleb formation Subjects were grouped according to BP recorded is likely indicative of apoptosis, thus this observa- during a screening visit; normal BP (N ¼ 9) o120/ tion adds to previous observations of increased 80 mm Hg (mean±s.d. systolic BP (SBP) ¼ 117.1± apoptosis in various tissues in hypertension. 6.7 mm Hg and diastolic BP (DBP) ¼ 70.2± Chronically elevated blood pressure (BP) is asso- 5.3 mm Hg); elevated BP (N ¼ 13) 4120/80 mm Hg ciated with a growing list of inflammatory markers (mean±s.d. SBP ¼ 142.5±8.2 mm Hg and DBP ¼ at rest and with exaggerated inflammatory responses 86.5±10.1 mm Hg). Subjects completed a VO2peak 1,2 to acute stress. Animal models of hypertension treadmill test and, approximately 1 week after the show that, in addition to other markers of leukocyte peak exercise test, returned for a 20-min steady-state activation, pseudopod projections are upregulated exercise task (65–70% VO2peak). A blood sample was alongside elevated BP. Pseudopods are temporary collected before (baseline) and during the last projections formed through F-actin polymerization minute of exercise. used by cells in phagocytosis and spreading and Blood was drawn into tubes containing sodium migration.3 Pseudopod formation may be important heparin, and immediately (within 30 s) 500 ml was in BP control because of its contribution to vascular removed and added to 500 ml 1% formaldehyde. resistance in the microvasculature, especially in After fixation for 410 min, red blood cells were capillaries, which demand single-file cell motion. lysed, and cells were washed and collected by Owing to their relatively large size, leukocyte centrifugation. Cells were resuspended in 25 ml velocity is reduced in capillaries, and thus con- phosphate-buffered saline and stained with crystal tributes to increase vascular viscosity and micro- violet for identification of nuclear morphology. vascular resistance. Un-fixed, whole blood samples were incubated with In addition to pseudopod projections, leukocytes phosphate-buffered saline as the negative control also project membrane blebs, typically in response and N-formyl-met-leu-phe (Sigma-Aldrich, St Louis, to toxic stimuli, and their appearance precedes MO, USA) as the positive control (100 ml for 5 min) apoptosis.4 Blebs form when the cytoskeleton and and then fixed with formaldehyde and followed the membrane separate.5 Transient, spherical bleb mem- same protocol. A light microscope was used to visualize brane extensions are seen during mitosis or during the cells (  100) and photographs of 70 cells per spreading in eukaryotic cells, but much larger blebs sample were acquired using a  20 eyepiece camera that do not shrink are found during the early (MiniVID, LW Scientific, Inc, Lawrenceville, GA, USA). apoptotic process due to loss of membrane integrity. Two blinded observers analysed images of each Physical and chemical stress have been shown to cell for pseudopod and bleb formation. Membrane induce apoptotic blebbing, and recently, neutrophils blebs were notable in a high proportion of samples. were observed to form large membrane blebs as a Cells were classified positive for pseudopod forma- precursor to necrosis after exposure to cytotoxic tion when projections were 41 mm in length. Cells factors.6 were classified positive for bleb formation when The current study investigated the effects of BP on projection was spherical and smooth and unstained the physical properties of circulating leukocytes at by crystal violet (see Figure 1 insert). Cells observed rest and in response to a moderate exercise chal- were identified by morphology, 490% were neu- lenge in otherwise healthy untreated individuals trophils. Inter- and intra-observer variation in bleb with a range of normal to elevated BP. We hypothe- and pseudopod classification was o4 and o2%, sized that pseudopod formation would be greater in respectively. Positive controls showed 94–98% Research Letter 685 subjects with elevated BP showed a significantly 60 greater proportion of cells with bleb formation (elevated BP ¼ 33.0±13%, normal BP ¼ 13.5±8%, 50 P ¼ 0.001). After exercise, there was a trend towards greater bleb appearance in cells from elevated BP 40 subjects (elevated BP ¼ 35.2±20%, normal BP ¼ 22.3±11%, P ¼ 0.09). Figure 1 demonstrates 30 the significant positive associations between pro- portion of cells with blebs at baseline with resting 20 SBP (r ¼ 0.623, P ¼ 0.002) and resting DBP (r ¼ 0.553,

% Cells with Blebs 10 P ¼ 0.008). Further, proportion of cells with blebs after exercise was also associated with both resting 0 SBP (r ¼ 0.471, P ¼ 0.027) and resting DBP (r ¼ 0.584, P ¼ 0.004). The current findings show that circulating leuko- 100 110 120 130 140 150 160 cytes in individuals with elevated BP as well as Resting SBP (mmHg) normal BP exhibit low levels of pseudopod projec- 60 tion before and even after acute exercise (o4%). Activation of cells in the circulation rapidly leads to 50 entrapment in capillaries as well as tethering and migration through the endothelium, and thus re- 40 moval from the active peripheral circulation, which could lead to low levels of activation in the 30 circulating pool of leukocytes drawn in whole blood. In vivo analysis in spontaneously hyperten- 20 sive rats has shown increased pseudopod formation 3 % Cells with Blebs 10 in capillaries, but the circulating cells analysed in the current data from venous blood did not exhibit 0 the same effect. The fixation protocol using formaldehyde resulted in prominent bleb formation on leukocytes. The 50 60 70 80 90 100 110 blebs observed were large in size, and given the Resting DBP (mmHg) peripheral circulation origin of the cells, unlikely to Figure 1 Association between resting blood pressure (SBP upper be formed in locomotion. The fixation protocol thus panel, DBP lower panel) and percentage of cells showing bleb may have initiated apoptosis while fixing the cells, formation before exercise. Insert: B, bleb morphology; PP, capturing the bleb formation. Alternatively, it may pseudopod morphology. reflect a weaker binding of the membrane to the underlying cytoskeleton, potentially reflective of pseudopod formation, and negative controls showed pre-existing cellular damage. It is noteworthy that o4% pseudopod formation. cells from subjects with elevated BP were more Data were analysed by analysis of variance and susceptible to bleb formation after fixation than cells Pearson’s correlations. Statistical significance was from subjects with normal BP. These findings are considered at Po0.05; Z2 was determined as a consistent with previous animal studies, in which measure of effect size. Apart from the expected hypertension has been associated with enhanced differences in resting and exercise BP, individuals apoptosis in several different cell types in the with normal BP were not different from those with spontaneously hypertensive rats,7 including the elevated BP in age, body mass index or VO2peak, nor microvascular endothelium as well as in high- blood glucose or cholesterol. During a 20-min pressure regions of the vasculature. In human exercise, the two BP groups were not different for hypertension, circulating endothelial cells have 8 average heart rate, VO2, respiratory exchange ratio been noted (circulating endothelial cells have been or metabolic equivalents, indicating that all partici- found to be up to 90% apoptotic in other diseases), pants exercised at relatively the same intensity as and apoptosis is increased in endothelial cells in intended. atherosclerosis.9,10 There was a trend towards greater pseudopod Several limitations of this study need to be formation after exercise in cells from hypertensive acknowledged. First, our sample size was modest; participants (pre-exercise elevated BP ¼ 3.2±7%, however, the difference between groups was con- normal BP ¼ 2.3±5%, P ¼ 0.75, post-exercise ele- firmed by the continuous data analysis, confirming vated BP ¼ 3.3±4%, normal BP ¼ 0.7±1%, the finding. Second, the methodology was not P ¼ 0.07). Post-exercise pseudopod formation corre- specifically designed to induce bleb formation. lated with resting SBP (r ¼ 0.429, P ¼ 0.047) and However, formaldehyde has previously been found resting DBP (r ¼ 0.544, P ¼ 0.009). Before exercise, to increase the intracellular concentration of Ca2 þ

Journal of Human Hypertension Research Letter 686 and induce apoptosis,11 and Ca2 þ homeoestasis 1Department of Psychiatry, UCSD Medical Center, disturbance has been suggested as a major factor University of California, San Diego, 12 responsible for bleb formation. La Jolla, CA, USA; The current study demonstrates that leukocytes 2Department of Bioengineering, The Institute of from hypertensive individuals are more susceptible Engineering in Medicine, University of California, to membrane bleb formation, indicative of apopto- San Diego, La Jolla, CA, USA sis, than leukocytes from normotensive individuals. The association with BP was continuous: if split to E-mail: [email protected] normotensive (SBP o120 mm Hg), pre-hypertensive Published online 22 April 2010 (120 mm HgoSBP4140 mm Hg) and hypertensive (SBP 4140 mm Hg), bleb formation showed graded increases of 13, 27 and 32%, indicating that future References studies might explore the susceptibility of cells to bleb formation as an early marker of hypertension. 1 Edwards KM, Ziegler MG, Mills PJ. The potential anti- Regarding pseudopod formation, future studies inflammatory benefits of improving physical fitness in hypertension. J Hypertens 2007; 25(8): 1533–1542. could examine in vitro cell activation stimulating 2 Suematsu M, Suzuki H, Delano FA, Schmid-Schonbein naı¨ve cells with plasma from subjects with elevated GW. The inflammatory aspect of the microcirculation or normal BP, to avoid loss of activated leukocytes in hypertension: oxidative stress, leukocytes/endothe- due to entrapment in the microcirculation. lial interaction, apoptosis. Microcirculation 2002; 9(4): 259–276. 3 Fukuda S, Yasu T, Kobayashi N, Ikeda N, Schmid- Schonbein GW. Contribution of fluid shear response in What is known about topic leukocytes to hemodynamic resistance in the sponta- K Hypertension is associated with many different markers of neously hypertensive rat. Circ Res 2004; 95(1): elevated inflammation, including pro-inflammatory 100–108. cytokines, reactive oxygen species and markers of leukocyte 4 Fadeel B. Plasma membrane alterations during apop- activation. K Leukocytes from spontaneously hypertensive rats (SHRs) tosis: role in corpse clearance. Antioxid Redox Signal have been shown to exhibit increased pseudopod 2004; 6(2): 269–275. extension, which is hypothesized to contribute to 5 Dai J, Sheetz MP. Membrane tether formation from microvascular resistance and thus elevated blood pressure. blebbing cells. Biophys J 1999; 77(6): 3363–3370. K SHR models show increased apoptosis in various cell types. 6 Penn AH, Hugli TE, Schmid-Schonbein GW. Pancrea- tic enzymes generate cytotoxic mediators in the What this study adds intestine. Shock 2007; 27(3): 296–304. K We demonstrate that leukocytes from hypertensive 7 Lim HH, DeLano FA, Schmid-Schonbein GW. Life and individuals are more susceptible to form membrane blebs, death cell labeling in the microcirculation of the indicative of apoptosis, than leukocytes from normotensive individuals. spontaneously hypertensive rat. J Vasc Res 2001; K Bleb formation was strongly associated with resting blood 38(3): 228–236. pressure. 8 Hladovec J, Prerovsky I. Endothelial lesion in hyper- tension. Cor Vasa 1989; 31(1): 51–54. 9 Alvarez RJ, Gips SJ, Moldovan N, Wilhide CC, Milliken EE, Hoang AT et al. 17beta-estradiol inhibits apoptosis Conflict of interest of endothelial cells. Biochem Biophys Res Commun 1997; 237(2): 372–381. The authors declare no conflict of interest 10 Tricot O, Mallat Z, Heymes C, Belmin J, Leseche G, Tedgui A. Relation between endothelial cell apoptosis and blood flow direction in human atherosclerotic plaques. Circulation 2000; 101(21): 2450–2453. Acknowledgements 11 Oyama Y, Sakai H, Arata T, Okano Y, Akaike N, Sakai K This work was supported by Grants HL57265 and et al. Cytotoxic effects of methanol, formaldehyde, and HL073355 from the National Institutes of Health formate on dissociated rat thymocytes: a possibility of aspartame toxicity. Cell Biol Toxicol 2002; 18(1): and the UCSD General Clinical Research Center 43–50. (MO1RR-00827). 12 Jewell SA, Bellomo G, Thor H, Orrenius S, Smith M. Bleb formation in hepatocytes during drug metabolism KM Edwards1, B Sheu1,2, S Hong1, AH Penn2, is caused by disturbances in thiol and calcium ion GW Schmid-Scho¨nbein2 and PJ Mills1 homeostasis. Science 1982; 217(4566): 1257–1259.

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