Basic Sciences

Shear-Sensitive Regulation of Neutrophil Flow Behavior and Its Potential Impact on Microvascular Blood Flow Dysregulation in HypercholesterolemiaSignificance

Xiaoyan Zhang, Ran Cheng, Dylan Rowe, Palaniappan Sethu, Alan Daugherty, Guoqiang Yu, Hainsworth Y. Shin
https://doi.org/10.1161/ATVBAHA.113.302868
Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:587-593
Originally published February 19, 2014

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Abstract

Objective—Shear stress–induced pseudopod retraction is an anti-inflammatory measure that minimizes neutrophil activity and is regulated by membrane cholesterol. We tested the hypothesis that a hypercholesterolemic impairment of shear mechanotransduction alters the neutrophil flow behavior leading to microvascular dysfunction.

Approach and Results—We examined the shear effects on the flow behavior of human leukocytes. When subjected to shearing during cone-plate viscometry, leukocyte suspensions exhibited parallel time-dependent reductions in viscosity and pseudopod activity. Shear-induced reductions in suspension viscosity were attenuated by membrane cholesterol enrichment. We also showed that enhanced pseudopod activity of leukocyte suspensions in 10% hematocrit significantly (P<0.05) raised the flow resistance of microvascular mimics. These results implicate an impaired neutrophil pseudopod retraction response to shear in hypercholesterolemic microvascular dysfunction. We confirmed this using near-infrared diffuse correlation spectroscopy to assess skeletal muscle blood flow regulation in the hindlimbs of mice subjected to reactive hyperemia. Using a custom protocol for the mouse, we extrapolated an adjusted peak flow and time to adjusted peak flow to quantify the early phase of the blood flow recovery response during reactive hyperemia when shear mechanobiology likely has a maximal impact. Compared with mice on normal diet, hypercholesterolemic mice exhibited significantly (P<0.05) reduced adjusted peak flow and prolonged time to adjusted peak flow which correlated (r=0.4 and r=−0.3, respectively) with neutrophil shear responsiveness and were abrogated by neutropenia.

Conclusions—These results provide the first evidence that the neutrophils contribute to tissue blood flow autoregulation. Moreover, a deficit in the neutrophil responsiveness to shear may be a feature of hypercholesterolemia-related microvascular dysfunction.

Introduction

Although hypercholesterolemia is a major risk factor for heart disease and stroke,1 the link between plasma cholesterol levels and incidence of cardiovascular pathobiology is not without controversy.2,3 In addition to its pathological effects on large vessels, hypercholesterolemia promotes microvascular dysfunction.4,5 Efforts to link microvascular dysfunction to hypercholesterolemia focused on its detrimental impact on the endothelial regulation of arteriolar vasoactivity.6,7 However, endothelium-mediated vasomotor control is not the sole determinant of tissue perfusion. Other factors that impact hemodynamic resistance are plasma lipid concentrations8 and blood cell rheology, both of which affect blood viscosity.8,9

Neutrophils, although having little effect on large vessel blood flow, influence blood rheology in the microcirculation where vessel diameters (4–100 μm) are similar to their cell dimensions.9–11 Once activated, neutrophils project pseudopods and bind to other cells, such as other leukocytes, endothelium, and platelets, all of which hinder their passage through the microvasculature and raise peripheral resistance.11,12 Thus, neutrophil activation profoundly affects microvascular flow.

Under physiological conditions, neutrophil activity is restricted by various anti-inflammatory factors. Among these, there is compelling evidence10 from human and rodent studies that shear stress is anti-inflammatory for neutrophils. Specifically, acute exposure of neutrophils to shear reduces their pseudopod activity, F-actin content, and surface expression of CD18 integrins.10 By doing so, shear stress mechanotransduction serves as a control mechanism that ensures neutrophils adopt a rounded, deformable, and nonadhesive state so as to minimize their impact on peripheral resistance. This possibility is in line with reports13,14 that an impaired neutrophil shear response raises microvascular resistance.

Notably, an impaired control of neutrophil pseudopod activity by shear stress develops early during development of a hypercholesterolemic blood state in mice fed a high-fat diet (HFD).15 Considering the link between neutrophil pseudopod activity and tissue blood flow, we hypothesized that a deficit in membrane cholesterol-related regulation of neutrophils by shear contributes to microvascular dysfunction in hypercholesterolemia.

We used real-time viscometry of leukocyte suspensions and microfluidics to link microvessel resistance to shear regulation of neutrophil flow behavior. We also used low-density lipoprotein receptor–deficient mice fed a normal diet (ND) or HFD to reveal a first correlative link between neutrophil shear sensitivity and in vivo tissue blood flow regulation. For this purpose, we tested the perfusion recovery responses of the posterior thigh muscles of mice subjected to transient blood flow occlusion (ie, reactive hyperemia [RH]). Conceivably, neutrophils in tissues undergoing RH experience a no-flow situation that mildly activates them16 because of upstream blood flow occlusion followed by an acute exposure to shear during reperfusion. This scenario implicates neutrophil shear sensitivity as a component of RH.

To relate changes in tissue blood flow autoregulation to altered neutrophil shear sensitivity, we used a novel optical technology: near-infrared diffuse correlation spectroscopy (DCS).17 This technique provided noninvasive, real-time tissue blood flow measurements deep in the murine thigh muscle to detect the neutrophil impact on the in vivo dynamics of RH. The combined use of classical cell biomechanics methods, current microfluidics approaches, and state-of-the-art optical spectroscopy revealed novel mechanistic insight regarding hypercholesterolemic microvascular dysfunction and the neutrophil.

Materials and Methods

Materials and Methods are available in the online-only Supplement.

Results

Shear-Induced Pseudopod Retraction Impacted Flow Behavior of Neutrophils in Suspension

To assess effects of shear stress on leukocyte flow (ie, tumbling) behavior, the suspension viscosities of leukocyte-enriched plasma, mildly stimulated with 10 nmol/L f-Met-Leu-Phe, were examined during 10-minute exposure to cone-plate shear flow. At the initial time point (t=30 seconds after flow onset), leukocyte-enriched plasma exhibited significantly (P<0.05) higher viscosities compared with cell-free plasma (Figure 1A). Although viscosities of cell-free plasma remained constant, viscosities of leukocyte-enriched plasma decreased in a time-dependent manner during viscometry (Figure 1A). After 9 minutes, viscosities of leukocyte-enriched plasma were reduced to levels similar to those of cell-free plasma.

Figure 1.
Figure 1.

Time-dependent reductions in cell suspension viscosity reflected shear-induced pseudopod retraction. Leukocyte-enriched plasma diluted 1:10 vol/vol in buffer was stimulated with 10 nmol/L f-Met-Leu-Phe and subjected to cone-plate viscometry (shear rate: 450/s or shear stress: ≈5 dyn/cm2). Instantaneous viscosities of cell suspensions (A) and time course of pseudopod activity and neutrophil-platelet adhesion (NPA) during a 10-minute duration (B) were examined. Data are mean±SEM from n=4 experiments. #P<0.05 compared with cell-free plasma at each time point using Student t test. * and +P<0.05 compared with t=30 seconds or 0 using 1-way repeated measures or regular ANOVA with Dunnett’s method.

In separate experiments, the percentage of neutrophils in leukocyte-enriched plasma that displayed pseudopods (Figure I in the online-only Data Supplement) decreased in a time-dependent fashion under shear with significant (P<0.05) reductions detected after 1 minute of flow (Figure 1B). In contrast, the percentage of neutrophils with bound platelets in these cell populations increased under shear and plateaued after 5 minutes of flow at levels significantly (P<0.05) higher than those observed at the initial time point (Figure 1B). Moreover, cysteine protease inhibitor, E64, that blocks CD18 cleavage and enhances neutrophil-platelet binding under shear,18 had no effect on the apparent viscosity of f-Met-Leu-Phe–stimulated leukocyte-enriched plasma throughout the duration of cone-plate viscometry. Viscosities of these cell suspensions were significantly (P<0.05) reduced after 2 minutes of shear in both the absence and presence of E64, relative to their initial viscosities at t=30 seconds (Figure 2).

Figure 2.
Figure 2.

Shear-induced reductions in cell suspension viscosity occurred independently of CD18-mediated neutrophil-platelet binding. Leukocyte-enriched plasma diluted 1:10 v/v in buffer was stimulated with 10 nmol/L f-Met-Leu-Phe in the absence (untreated [UT]) or presence of 28 μmol/L E64 and then subjected to cone-plate viscometry (shear rate: 450/s). Instantaneous viscosities of cell suspensions were examined and normalized to their initial viscosity at t=30 seconds. Data are mean±SEM from n=3 experiments. * and +P<0.05 compared with t=30 seconds using 1-way repeated measures ANOVA with Dunnett’s method.

Cell Membrane Cholesterol Enrichment Altered Shear Stress Influence on Leukocyte Rheology

Incubation of leukocytes with cholesterol:methyl-β-cyclo dextrin complexes (CH) for all concentrations tested had no effect on their initial suspension viscosity at 30 seconds after flow onset (data not shown). Beyond this 30-second time point, membrane cholesterol–enhancing agents dose dependently impaired shear-related reductions in leukocyte suspension viscosity (Figure 3). Specifically, leukocyte suspensions incubated with 0, 2, or 5 μg/mL CH exhibited significantly (P<0.05) reduced viscosities after 4 minutes of cone-plate flow relative to their initial viscosities at t=30 seconds. However, viscosity reductions for leukocyte-enriched plasma incubated with either 2 or 5 μg/mL CH were smaller relative to those for naïve cells. Moreover, viscosities of leukocyte-enriched plasma incubated with 10 μg/mL CH remained the same for the duration of viscometry. The dose-dependent effect of membrane cholesterol enhancement was confirmed by linear regression analyses of the end point (ie, t=10 minutes) viscosity measurements versus CH concentration. We detected a significant correlation (R2=0.94; P<0.05) between leukocyte suspension viscosity and CH concentration.

Figure 3.
Figure 3.

Membrane cholesterol enrichment altered shear-sensitive leukocyte rheological flow behavior. Leukocytes enriched with membrane cholesterol by incubation in 0 to 10 μg/mL cholesterol:methyl-β-cyclodextrin complexes (CH) were stimulated with 10 nmol/L f-Met-Leu-Phe and subjected to viscometry (shear rate: 450/s). A, Instantaneous viscosities of cell suspensions were monitored and normalized to the initial values at t=30 seconds. B, The end point viscosities after 10-minute shear exposure were correlated with CH concentration. Data are mean±SEM from n≥5 experiments. *, +, and #P<0.05 compared with t=30 seconds using 1-way repeated measures ANOVA with Dunnett’s method.

Pseudopod Projection Influenced Resistance of a Microfluidics-Based Microvascular Mimic to Flow of Neutrophil Suspensions in the Presence of Red Blood Cells

We confirmed, using microfluidics, that the impact of neutrophil pseudopod projection on suspension viscosity influenced microvessel flow resistance. On injection of suspensions of purified neutrophils through a 50×500 µm microchannel at a constant flow of 1 mL/h, the pressure difference across the microfluidic channel was similar for perfusate containing either activated or inactivated cells (Figure 4A). Accordingly, there was no difference in microchannel flow resistance imposed by these 2 types of cell suspensions (Figure 4B). In the presence of 10% hematocrit, perfusion of activated neutrophils enhanced pressure difference across the microchannel (Figure 4A) resulting in significant (P<0.05) elevations (17.5%) in microchannel resistance relative to perfusion of nonactivated cells (Figure 4B). Separate experiments confirmed that the percentage of neutrophils with pseudopod(s) was significantly (P<0.05) enhanced by stimulation with 10 nmol/L f-Met-Leu-Phe (Figure 4C and 4D).

Figure 4.
Figure 4.

Perfusion with activated neutrophils increased microchannel resistance in the presence of red blood cells. A and B, Purified neutrophils (PMNs) were activated by 10 nmol/L f-Met-Leu-Phe (fMLP), fixed, and then perfused through 500 µm (w)×50 µm (h)×20 mm (l) microfluidics-based microvascular mimics at 1 mL/h in the absence or presence of 10% hematocrit (Hem). Controls were unstimulated cells. Pressure differences across the microchannel were recorded (A), and flow resistance was calculated (B). C and D, Pseudopod activity of unstimulated PMNs and fMLP-activated cells were examined under microscope (C), and the percentage of cells with pseudopods was counted (D). Data in B and D are mean±SEM from n=4 experiments. *P<0.05 using Student t test.

Neutrophils Contributed to Hypercholesterolemia-Induced Microvascular Dysregulation

The blood flow recovery responses of hindlimbs of ND- and HFD-fed mice to 5-minute blood flow occlusion were assessed to explore the neutrophil contribution to hypercholesterolemic microvascular dysfunction. Compared with their ND-fed counterparts, low-density lipoprotein receptor–deficient mice fed a HFD exhibited significant (P<0.05) time-dependent increases in plasma concentrations of free and total cholesterol (Table I in the online-only Data Supplement). For RH analyses, we ensured that during cuff occlusion, relative changes of blood flow (rBF) values were <10% relative to preocclusion levels for all animals tested (Table II in the online-only Data Supplement). We previously reported that successful blood flow restriction is repeatedly and reliably achieved once rBF, during cuff occlusion, reached <10% of its preocclusion value.17

Notably, the rBF curves recorded for the posterior thigh muscles subjected to blood flow occlusion displayed a transient flow overshoot after cuff release followed by a return to baseline levels within 30 minutes (Figure 5A–5C). To quantify RH, all rBF measurements fluctuating within 10% of the peak flow value were averaged and defined as adjusted peak flow (APF). Using this approach, we did not observe differences in rBF (data not shown) or APF (Figure 5D) between mice on HFD or ND for 2 and 4 weeks. In contrast, rBF curves for mice fed a HFD for 8 weeks displayed a blunted peak overshoot compared with that of their ND-fed counterparts (Figure 5B). This blunting of rBF curves for 8-week HFD-fed mice manifested as significant (P<0.05) reductions in APF relative to that for their ND-fed counterparts (Figure 5D). Markedly, acute depletion of ≈90% neutrophils from mice abrogated the effects of 8-week HFD on peak rBF and APF (Figure 5C and 5E).

Figure 5.
Figure 5.

Neutrophils contributed to hypercholesterolemia-induced impairment of reactive hyperemia. A, A representative diffuse correlation spectroscopy flow curve demonstrated relative changes of blood flow (rBF) before, during, and after 5-minute cuff occlusion. Adjusted peak flow (APF) was defined as the mean of maximal 10% of rBF data points (solid black circles in curve). Time to APF (TAPF) was defined as the mean of corresponding time points for maximal 10% of rBF data points (solid black circles in curve). B and C, Two representative rBF curves were overlaid for regular (B) and neutropenic (C) mice fed normal diet (ND) or high-fat diet (HFD) for 8 weeks. D and E, APF values were calculated for regular (D; n=7) and neutropenic (E; n=6) mice on ND or HFD. Data in D and E are mean±SEM. *P<0.05; 2-way ANOVA detected diet had effects, then Student t test was performed between ND and HFD at each time point. F, TAPF for all animals tested was displayed in a box-and-whisker plot. Whiskers were the minimum and maximum values; median values were indicated by horizontal lines in boxes; n=7 for regular mice experiments and n=6 for neutropenia experiments. #P<0.05 using Mann–Whitney U test.

To determine the temporal effects of neutrophils on RH, time points corresponding to the rBF measurements used to calculate APF were averaged and defined as time to APF (TAPF). Overall, median values of TAPF for ND-fed mice fell between 1 and 2 minutes after cuff release (Figure 5F). Notably, HFD-fed mice exhibited diet duration–dependent delays in TAPF relative to ND-fed mice. Mice on either ND or HFD for 2 and 4 weeks exhibited similar TAPF. However, after 8 weeks of diet, TAPF was significantly (P<0.05) longer for HFD-fed mice compared with that for their ND-fed counterparts (Figure 5F). Again, after acute induction of neutropenia, TAPF was similar for mice fed either an ND or HFD for 8 weeks (Figure 5F).

Finally, although the pseudopod activity levels of neutrophils maintained under static (no flow) conditions after blood draw were unaffected by diet type and duration (Table III in the online-only Data Supplement), HFD altered shear responses (ie, pseudopod retraction) of these cells in a diet duration-dependent fashion. Neutrophils from ND-fed mice for all diet durations exhibited significant (P<0.05) reductions in pseudopod activity in response to in vitro exposure to 5 dyn/cm2 for 10 minutes (Table IV in the online-only Data Supplement). Neutrophils from mice subjected to HFD for 2 and 4 weeks exhibited no reductions in pseudopod activity after shear exposure; their shear response indexes approached 0 (Table IV in the online-only Data Supplement). For mice subjected to HFD for 8 weeks, their neutrophils exhibited reversed shear responses with indices that were significantly (P<0.05) <0, that is, pseudopod extension in response to shear (Table IV in the online-only Data Supplement).

Based on Pearson correlation analyses, the neutrophil shear response index, APF, and TAPF from all mice significantly (P<0.05) correlated with serum concentrations of total and free cholesterol (Table). There were also significant (P<0.05) correlations between the neutrophil shear index and either APF or TAPF (Table).

View this table:
Table.

Pearson Correlation Analyses of the Impact of Hypercholesterolemia and the Neutrophil Shear Responsiveness on Postocclusive Reactive Hyperemia

Discussion

Pseudopod formation enhances the tumbling of neutrophils in the parabolic velocity field of blood flow in microvessels and promotes their collisions with red blood cells. These collisions displace red blood cells from their axial position and into the peripheral cell-free plasma layer typical of blood flow in microvessels with an adverse effect on blood viscosity.9,11 Neutrophil homotypic or heterotypic binding (eg, to platelets) may also amplify these rheological effects.19

In contrast, we showed that shear-induced pseudopod retraction minimizes neutrophil tumbling. Conceivably, prestimulated neutrophils in the linear velocity gradient of cone-plate flow retracted their pseudopods, became rounded, and likely reduced their cell–cell collisions (Figure II in the online-only Data Supplement). The neutrophils were likely the major contributors to this effect because of their large numbers in plasma (≈60%–70% of the leukocrit). Monocytes are present in low numbers in plasma (≈5% of the leukocrit) and thus likely had a negligible impact. Lymphocytes did not extend pseudopods and thus did not exhibit shear-related retraction (data not shown). Finally, because cell shape changes attributable to bound platelets were likely smaller than those attributable to pseudopods,20 it was not surprising that platelet binding had no effect.

We reported15 that membrane cholesterol loading, using CH, dose dependently impairs shear-induced pseudopod retraction. This cholesterol effect on the neutrophils translates to an impact on their flow behavior. Reportedly, 90% of free in the cell cholesterol resides in the peripheral membrane, suggesting that effects of CH likely resulted from its rigidifying actions on this lipid structure.10 It is possible that CH altered cholesterol distribution among the cytosolic organelles or affected the activity of cell surface proteins because of direct hydrophobic interactions.21 However, we showed previously15 that a dose-dependent blockade of neutrophil shear responses by CH results from changes in neutrophil membrane fluidity.

Notably, hypercholesterolemia raises membrane cholesterol content, and reduces membrane fluidity, of leukocytes in blood.22 Such findings, combined with our data, point to a putative link between blood cholesterol elevations and impaired neutrophil shear regulation. However, the in vitro effects of CH on neutrophil membranes may differ from that due to in vivo blood cholesterol elevations. Low-density lipoprotein particles, the main in vivo cholesterol carriers, may deliver cholesterol into leukocyte membranes less efficiently than CH. Other factors arising from hypercholesterolemia may also alter neutrophil shear sensitivity including changes in plasma composition (eg, inflammatory agonists, proteases) and the activity of other vascular cells.10

Despite this, the importance of the neutrophil shear response is evident considering that pseudopod formation by flowing leukocytes raises peripheral resistance as previously reported23,24 and in line with our own microfluidics data. Consistent with prior studies,9,11,25 we showed that hydrodynamic leukocyte–erythrocyte interactions, and not cell activation alone, are responsible for their impact on flow resistance. The ability of shear to reduce neutrophil pseudopod activity likely minimizes such hydrodynamic interactions and, thus, microvessel flow resistance. Previous in vivo studies have shown that impaired neutrophil pseudopod retraction responses to shear elevate peripheral resistance.13,14 But these results point to a passive effect.

Control of neutrophil shear sensitivity may, in fact, contribute to microvascular control of peripheral resistance and tissue blood flow. To explore this possibility, we assessed the neutrophil impact on RH, which is impaired by hypercholesterolemia.6,26 Recently, we developed a procedure27 to use DCS to monitor, in real time, rBF in muscles deep (3 mm below the skin) in the thigh of low-density lipoprotein receptor–deficient mice subjected to RH.27 DCS has been validated directly in many murine and human tissues against laser Doppler, Doppler ultrasound, Xenon-computed tomography, microsphere velocimetry, power spectral ultrasound, and arterial spin–labeled MRI.17,28–36 Moreover, DCS for RH in our mice provided similar blood flow data27 to that reported for MNRI-C57BL/6 mice using arterial spin–labeled MRI.29 The higher time resolution of DCS, however, allowed us to relate the kinetics of neutrophil shear responses to RH.

Using DCS, we showed that neutrophils (1) play a role in RH and (2) contribute to dysregulated RH during hypercholesterolemia. Notably, the neutropenia data implied that lymphocytes had no impact on RH. A role for monocytes also seemed to be excluded despite reports that their numbers in blood increase during hypercholesterolemia.37 Although other Ly6G-positive cells, such as eosinophils and dendritic cells, in blood may affect RH,38 their low numbers, relative to neutrophils, likely made their impact small. But cell number is not the main factor because reductions in neutrophil numbers alone did not account for the neutropenic effect on RH in HFD-fed mice. In fact, peripheral resistance is independent of leukocyte concentrations between 3.6×106/mL and 6.2×106/mL in blood.11

In addition to neutrophilia, hypercholesterolemia promotes accumulation of activated neutrophils in the microvasculature that perpetuates a chronic inflammatory state and indirectly impacts peripheral resistance via downstream effects on arteriolar endothelium.39 Our results substantiate a neutrophil role in hypercholesterolemic microvascular pathobiology.10 Specifically, we provide correlative evidence suggesting that in addition to releasing inflammatory agonists, activated neutrophils with impaired shear responses attributable to pathological blood cholesterol elevations also promote microvascular dysfunction by physically disturbing blood flow.

Markedly, time-dependent impairment of neutrophil shear sensitivity long preceded microvascular dysfunction as detected using our novel blood flow indices (ie, APF and TAPF). Moreover, the neutrophil impact on RH in our mice largely occurred rapidly after cuff release that tracked with the temporal kinetics of shear-induced pseudopod retraction as defined by our in vitro studies. Thus, APF and TAPF seem to be sensitive to shear-related neutrophil contributions to RH. In addition, DCS was able to detect these contributions.

However, the link between either APF or TAPF and neutrophil shear responsiveness reflects a correlative, and not a cause–effect, relationship. Notably, this link was not attributable to differences in baseline pseudopod activity of neutrophils in blood from ND-fed and HFD-fed mice (Table III in the online-only Data Supplement). Although our baseline indices may not represent the instantaneous activity state of neutrophils in vivo, there is no evidence, to our knowledge, that their morphology is altered because of hypercholesterolemia. Finally, the actions of other leukocyte subtypes, platelets, and endothelium on neutrophil activity, for example, via release of cell agonists,40 may have contributed to the hypercholesterolemic impact on RH. But despite these possibilities, the neutrophil seems to be the key player, as verified by our neutropenia results.

Moreover, the correlation between shear sensitivity and RH did not account for neutrophil adhesion in the microcirculation, which can dramatically raise hemodynamic resistance by reducing microvascular radii.11 Shear stress is antiadhesive for neutrophils by promoting cleavage of cell surface CD18 integrins.10,18 Notably, cholesterol influences the regulation of CD18-related neutrophil adhesivity.41,42 Its enrichment in the cell membrane raises CD18 surface levels and neutrophil adhesion.42,43 Thus, the major impact of hypercholesterolemia on RH may, in fact, be attributable to its effects on shear-sensitive CD18 proteolysis. Despite this, our data are still the first to implicate neutrophil shear sensitivity in RH.

Considering that microvascular dysfunction forecasts hypercholesterolemic vasculopathy,4,5 impaired neutrophil shear sensitivity upstream of dysregulated RH may be an early symptom of the harmful impact of high blood cholesterol. Thus, ex vivo blood cell measures and in vivo tissue blood flow indices that account for neutrophil shear sensitivity may be prognostic, and not just diagnostic, of hypercholesterolemia-related pathobiology. Our results also point to a potential strategy to target a source of microvascular dysfunction attributable to hypercholesterolemia. Specifically, because acutely fluidizing the membranes of neutrophils counteracts the effects of excess cholesterol on their shear responsiveness,15 membrane fluidizers may be used to ameliorate microvascular dysfunction attributable to hypercholesterolemia while administering cholesterol-lowering drugs during the long term. The potential benefit of membrane fluidizers on hypercholesterolemic microvasculature has been supported by evidence from other investigators.43 In these ways, the insight revealed by this study may serve as the basis for the design of new clinical approaches focused on the pathobiology, and not just on cholesterol levels, something that has come into question as the most effective strategy to treat hypercholesterolemic vascular disease.

Acknowledgments

We thank Dr Kimberly Anderson for the use of the oxygen plasma deposition system as well as Dr Stephen Lai-Fook and Dr Eugene Bruce for their technical and material assistance.

Sources of Funding

This work was supported by an American Heart Association Beginning-Grant-in-Aid and a National Science Foundation-Kentucky Experimental Program to Stimulate Competitive Research, Bioen gineering Initiative Grant.

Disclosures

None.

Footnotes

  • ↵* X. Zhang and R. Cheng shared first authorship.

  • This manuscript was sent to Theo van Berkel, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.302868/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    APF
    adjusted peak flow
    CH
    cholesterol:methyl-β-cyclodextrin complexes
    DCS
    diffuse correlation spectroscopy
    HFD
    high-fat diet
    ND
    normal diet
    rBF
    relative changes of blood flow
    RH
    reactive hyperemia
    TAPF
    time to adjusted peak flow

  • Received June 25, 2013.
  • Accepted January 13, 2014.

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Significance

Neutrophils have largely been neglected in hypercholesterolemia-related pathobiology. We integrated the use of cell mechanics with microfluidics methodologies to link cholesterol-related control of neutrophil pseudopod activity by shear stress to microvessel flow resistance. Moreover, using novel measures derived from a noninvasive, diffuse correlation spectroscopic technique, we revealed a first correlation linking hypercholesterolemic microvascular dysregulation, neutrophils and their impaired shear sensitivity. These results provide new insight regarding the link between hypercholesterolemia and microvascular dysfunction. Microvascular dysfunction long precedes, and is proposed to forecast, lethal hypercholesterolemic vasculopathy. In light of this, our study suggests that impaired neutrophil shear sensitivity is one of the earliest symptoms of the hypercholesterolemic impact on the microcirculation. As such, our results may serve as the basis for novel diagnostic strategies and therapeutic (eg, membrane fluidizing adjuvant) approaches that target an early symptom (eg, impaired neutrophil shear regulation) of hypercholesterolemic pathobiology other than cholesterol levels.

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  • Shear-Sensitive Regulation of Neutrophil Flow Behavior and Its Potential Impact on Microvascular Blood Flow Dysregulation in HypercholesterolemiaSignificance
    Xiaoyan Zhang, Ran Cheng, Dylan Rowe, Palaniappan Sethu, Alan Daugherty, Guoqiang Yu and Hainsworth Y. Shin
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:587-593, originally published February 19, 2014
    https://doi.org/10.1161/ATVBAHA.113.302868
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