The role of carotid plaque echogenicity in baroreflex sensitivity

ObjectiveThe baroreflex sensitivity is impaired in patients with carotid atherosclerosis. The purpose of our study was to assess the impact of carotid plaque echogenicity on the baroreflex function in patients with significant carotid atherosclerosis, who have not undergone carotid surgery.MethodSpontaneous baroreflex sensitivity (sBRS) was estimated in 45 patients with at least a severe carotid stenosis (70%-99%). sBRS calculation was performed noninvasively, with the spontaneous sequence method, based on indirectly estimated central blood pressures from radial recordings. This method failed in three patients due to poor-quality recordings, and eventually 42 patients were evaluated. After carotid duplex examination, carotid plaque echogenicity was graded from 1 to 4 according to Gray-Weale classification and the patients were divided into two groups: the echolucent group (grades 1 and 2) and the echogenic group (grades 3 and 4).ResultsSixteen patients (38%) and 26 patients (62%) were included in the echolucent and echogenic group, respectively. Diabetes mellitus was observed more frequently among echolucent plaques (χ2 = 8.0; P < .004), while those plaques were also more commonly symptomatic compared with echogenic atheromas (χ2 = 8.5; P < .003). Systolic arterial pressure, diastolic arterial pressure, and heart rate were similar in the two groups. Nevertheless, the mean value of baroreflex sensitivity was found to be significantly lower in the echogenic group (2.96 ms/mm Hg) compared with the echolucent one (5.0 ms/mm Hg), (F [1, 42] = 10.1; P < .003).ConclusionsThese findings suggest that echogenic plaques are associated with reduced baroreflex function compared with echolucent ones. Further investigation is warranted to define whether such an sBRS impairment could be responsible for cardiovascular morbidity associated with echogenic plaques

Spatiotemporal characterization of endothelial cell motility and physical forces during exposure to Borrelia burgdorferi

Cell motility and biomechanics are critical in various (patho)physiological processes, including the regulation of vascular barrier integrity, which can be subverted by bacterial pathogens. Here, we present a protocol on how to expose endothelial cells (ECs) to vector-borne Borrelia burgdorferi (Bb) and characterize EC kinematics and dynamics during exposure to live or heat-inactivated Bb through traction force and monolayer stress microscopy. Modifications to this protocol may be necessary for studying how different cell types interact with Bb or other microorganisms

A Stiff Extracellular Matrix Favors the Mechanical Cell Competition that Leads to Extrusion of Bacterially-Infected Epithelial Cells

Cell competition refers to the mechanism whereby less fit cells (“losers”) are sensed and eliminated by more fit neighboring cells (“winners”) and arises during many processes including intracellular bacterial infection. Extracellular matrix (ECM) stiffness can regulate important cellular functions, such as motility, by modulating the physical forces that cells transduce and could thus modulate the output of cellular competitions. Herein, we employ a computational model to investigate the previously overlooked role of ECM stiffness in modulating the forceful extrusion of infected “loser” cells by uninfected “winner” cells. We find that increasing ECM stiffness promotes the collective squeezing and subsequent extrusion of infected cells due to differential cell displacements and cellular force generation. Moreover, we discover that an increase in the ratio of uninfected to infected cell stiffness as well as a smaller infection focus size, independently promote squeezing of infected cells, and this phenomenon is more prominent on stiffer compared to softer matrices. Our experimental findings validate the computational predictions by demonstrating increased collective cell extrusion on stiff matrices and glass as opposed to softer matrices, which is associated with decreased bacterial spread in the basal cell monolayer in vitro. Collectively, our results suggest that ECM stiffness plays a major role in modulating the competition between infected and uninfected cells, with stiffer matrices promoting this battle through differential modulation of cell mechanics between the two cell populations

Borrelia burgdorferi modulates the physical forces and immunity signaling in endothelial cells

Borrelia burgdorferi (Bb), a vector-borne bacterial pathogen and the causative agent of Lyme disease, can spread to distant tissues in the human host by traveling in and through monolayers of endothelial cells (ECs) lining the vasculature. To examine whether Bb alters the physical forces of ECs to promote its dissemination, we exposed ECs to Bb and observed a sharp and transient increase in EC traction and intercellular forces, followed by a prolonged decrease in EC motility and physical forces. All variables returned to baseline at 24 h after exposure. RNA sequencing analysis revealed an upregulation of innate immune signaling pathways during early but not late Bb exposure. Exposure of ECs to heat-inactivated Bb recapitulated only the early weakening of EC mechanotransduction. The differential responses to live versus heat-inactivated Bb indicate a tight interplay between innate immune signaling and physical forces in host ECs and suggest their active modulation by Bb

The role of carotid plaque echogenicity in baroreflex sensitivity

Objective: The baroreflex sensitivity is impaired in patients with carotid atherosclerosis. The purpose of our study was to assess the impact of carotid plaque echogenicity on the baroreflex function in patients with significant carotid atherosclerosis, who have not undergone carotid surgery. Method: Spontaneous baroreflex sensitivity (sBRS) was estimated in 45 patients with at least a severe carotid stenosis (70%-99%). sBRS calculation was performed noninvasively, with the spontaneous sequence method, based on indirectly estimated central blood pressures from radial recordings. This method failed in three patients due to poor-quality recordings, and eventually 42 patients were evaluated. After carotid duplex examination, carotid plaque echogenicity was graded from 1 to 4 according to Gray-Weale classification and the patients were divided into two groups: the echolucent group (grades 1 and 2) and the echogenic group (grades 3 and 4). Results: Sixteen patients (38%) and 26 patients (62%) were included in the echolucent and echogenic group, respectively. Diabetes mellitus was observed more frequently among echolucent plaques (chi(2) = 8.0; P &lt; .004), while those plaques were also more commonly symptomatic compared with echogenic atheromas (chi(2) = 8.5; P &lt; .003). Systolic arterial pressure, diastolic arterial pressure, and heart rate were similar in the two groups. Nevertheless, the mean value of baroreflex sensitivity was found to be significantly lower in the echogenic group (2.96 ms/mm Hg) compared with the echolucent one (5.0 ms/mm Hg), (F[1, 42] = 10.1; P &lt; .003). Conclusions:These findings suggest that echogenic plaques are associated with reduced baroreflex function compared with echolucent ones. Further investigation is warranted to define whether such an sBRS impairment could be responsible for cardiovascular morbidity associated with echogenic plaques. (J Vasc Surg 2011;54:93-99.

<i>Listeria monocytogenes</i> InlP interacts with afadin and facilitates basement membrane crossing

<div><p>During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as <i>Listeria monocytogenes</i>, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the <i>L</i>. <i>monocytogenes</i> protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar <i>L</i>. <i>monocytogenes</i> internalin Lmo2027, binds to human afadin (encoded by <i>AF-6</i>), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca²⁺-binding site. Despite afadin’s involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by <i>AF-6</i> knock-out MDCK cells. <i>L</i>. <i>monocytogenes ΔinlP</i> mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in <i>inlP</i> (<i>inlP Δ</i>LRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote <i>L</i>. <i>monocytogenes</i> transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.</p></div

Bacterial effector InlP binds host adaptor protein afadin.

<p>(A-B) InlP-GST fusion protein (InlP) or GST protein alone (-) bound to glutathione-Sepharose resin were incubated overnight with protein extracts from (A) MDCK or MDCK <i>AF-6</i>^-/- cells or (B) human placenta. Input (IN) and elution fractions (EF) were analyzed by Western blot with anti-afadin antibodies. Actin: loading control. (C-E) Immunofluorescence of human placental villi stained for afadin (red) and DAPI (blue). In panel C the white bar = 10 μm, the asterisk labels a portion of the stroma, and the arrow points to some of the CTBs. White rectangles in panel C indicate the locations of zoomed insets shown in panels D-E.</p

LRR5 stabilized afadin- InlP interaction.

<p>(A) Loading control of InlP-afadin binding pull-down experiments with LRR mutants by Coomassie blue staining. GST protein alone (-), InlPΔLRR5-GST fusion protein (ΔLRR5), InlPΔLRR7-GST fusion protein (ΔLRR7), or InlP-GST (InlP) bound to glutathione-Sepharose resin were used as bait for pull-down experiments with protein extracts from MDCK cell line. Data shown are Coomassie staining, and the most abundant band in each lane represents the bait. (B-C) GST fusion proteins or GST protein alone (-) bound to glutathione-sepharose resin were incubated overnight with protein extracts from MDCK cells, and elution fractions (EF) were analyzed by Western blot with anti-afadin antibodies. The following GST fusion proteins were used: GST-InlP (InlP) and GST-InlP with deletions in LRR5 (ΔLRR5) or LRR7 (ΔLRR7). Actin: loading control. Undiluted elution fractions were analyzed with the exception of the elution fractions marked in panel C that were diluted 5-fold. Actin: loading control. (D) Scatter plot showing the logarithm of the ratio of intensity of InlP-GST bound proteins to the intensity of ΔLRR5-GST bound proteins versus total intensity of InlP-GST bound proteins. The intensities (A.U.) of the proteins were identified through mass spectrometry using InlP-GST or InlPΔLRR5-GST as baits to identify host binding partners in the MDCK cell culture extracts. Blue diamonds show all the proteins apart from afadin, which is indicated as orange square. Using this metric, afadin is 2.7 standard deviations away from the mean for this group.</p

InlP enhances transcytosis through MDCK polarized monolayers.

<p>(A) Intracellular growth curves of wild type 10403S <i>L</i>. <i>monocytogenes</i> in MDCK (black solid line) or MDCK <i>AF-6</i>^<i>-/-</i> (black dashed line) cells and of <i>ΔinlP L</i>. <i>monocytogenes</i> in MDCK (gray solid line) or MDCK <i>AF-6</i>^<i>-/-</i> (gray dashed line) cells. Unpolarized monolayers of MDCK cell lines were infected with 10403S at MOI of 150:1. Gentamicin was added at 50 μg/ml to kill extracellular bacteria and maintained in media thereafter. Each growth curve represents the means and standard deviations of colony forming units (CFU) over time from three separate experiments performed in triplicates. (B) Afadin limits initial <i>L</i>. <i>monocytogenes</i> invasion. Flow cytometry quantifying the number of <i>L</i>. <i>monocytogenes</i>-containing MDCK and MDCK <i>AF-6</i>^-/- cells 5 hours post infection. Data were normalized to 1 for wild-type MDCK cells infected with Δ<i>actA L</i>. <i>monocytogenes</i> for each experiment, and pooled from two independent experiments (each experiment is depicted by different symbols). (C) Amount of transcytosis by wild-type, Δ<i>inlP</i>, and Δ<i>actA L</i>. <i>monocytogenes</i> through wild-type MDCK monolayers. Data were normalized to 1 for MDCK cells infected with wild-type <i>L</i>. <i>monocytogenes</i> for each experiment, and pooled from four independent experiments. Each experiment is depicted by different symbols. (D) Amount of transcytosis by wild-type, Δ<i>inlP</i>, and Δ<i>actA L</i>. <i>monocytogenes</i> through MDCK <i>AF-6</i>^-/- monolayers. Data were normalized to 1 for <i>AF-6</i>^-/- MDCK cells infected with wild-type <i>L</i>. <i>monocytogenes</i> for each experiment, and pooled from three independent experiments. Each experiment is depicted by different symbols. (E) Amount of transcytosis by wild-type, Δ<i>inlP</i>, and ΔLRR5 <i>L</i>. <i>monocytogenes</i> through MDCK monolayers. Data were normalized to 1 for MDCK cells infected with wild-type <i>L</i>. <i>monocytogenes</i> for each experiment, and pooled from three independent experiments. Each experiment is depicted by different symbols.</p