Protective Role of the Podocyte IL-15 / STAT5 Pathway in Focal Segmental Glomerulosclerosis.

Introduction: During glomerular diseases, podocyte-specific pathways can modulate the intensity of histological disease and prognosis. The therapeutic targeting of these pathways could thus improve the management and prognosis of kidney diseases. The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway, classically described in immune cells, has been recently described in detail in intrinsic kidney cells. Methods: We describe STAT5 expression in human kidney biopsies from patients with focal segmental glomerulosclerosis (FSGS) and studied mice with a podocyte-specific Stat5 deletion in experimental glomerular diseases. Results: Here, we show, for the first time, that STAT5 is activated in human podocytes in FSGS. In addition, podocyte-specific Stat5 inactivation aggravates the structural and functional alterations in a mouse model of FSGS. This could be due, at least in part, to an inhibition of autophagic flux. Finally, interleukin 15 (IL-15), a classical activator of STAT5 in immune cells, increases STAT5 phosphorylation in human podocytes, and its administration alleviates glomerular injury in vivo by maintaining autophagic flux in podocytes. Conclusion: Activating podocyte STAT5 with commercially available IL-15 represents a potential new therapeutic avenue for FSGS.

Neuropilin-1 regulates renin synthesis in juxtaglomerular cells.

Renin is the key enzyme of the systemic renin-angiotensin-aldosterone system, which plays an essential role in regulating blood pressure and maintaining electrolyte and extracellular volume homeostasis. Renin is mainly produced and secreted by specialized juxtaglomerular (JG) cells in the kidney. In the present study, we report for the first time that the conserved transmembrane receptor neuropilin-1 (NRP1) participates in the development of JG cells and plays a key role in renin production. We used the myelin protein zero-Cre (P0-Cre) to abrogate Nrp1 constitutively in P0-Cre lineage-labelled cells of the kidney. We found that the P0-Cre precursor cells differentiate into renin-producing JG cells. We employed a lineage-tracing strategy combined with RNAscope quantification and metabolic studies to reveal a cell-autonomous role for NRP1 in JG cell function. Nrp1-deficient animals displayed abnormal levels of tissue renin expression and failed to adapt properly to a homeostatic challenge to sodium balance. These findings provide new insights into cell fate decisions and cellular plasticity operating in P0-Cre-expressing precursors and identify NRP1 as a novel key regulator of JG cell maturation. KEY POINTS: Renin is a centrepiece of the renin-angiotensin-aldosterone system and is produced by specialized juxtaglomerular cells (JG) of the kidney. Neuropilin-1 (NRP1) is a conserved membrane-bound receptor that regulates vascular and neuronal development, cancer aggressiveness and fibrosis progression. We used conditional mutagenesis and lineage tracing to show that NRP1 is expressed in JG cells where it regulates their function. Cell-specific Nrp1 knockout mice present with renin paucity in JG cells and struggle to adapt to a homeostatic challenge to sodium balance. The results support the versatility of renin-producing cells in the kidney and may open new avenues for therapeutic approaches.

Involvement of an IgE/Mast cell/B cell amplification loop in abdominal aortic aneurysm progression.

AIMS: IgE type immunoglobulins and their specific effector cells, mast cells (MCs), are associated with abdominal aortic aneurysm (AAA) progression. In parallel, immunoglobulin-producing B cells, organised in tertiary lymphoid organs (TLOs) within the aortic wall, have also been linked to aneurysmal progression. We aimed at investigating the potential role and mechanism linking local MCs, TLO B cells, and IgE production in aneurysmal progression. METHODS AND RESULTS: Through histological assays conducted on human surgical samples from AAA patients, we uncovered that activated MCs were enriched at sites of unhealed haematomas, due to subclinical aortic wall fissuring, in close proximity to adventitial IgE+ TLO B cells. Remarkably, in vitro the IgEs deriving from these samples enhanced MC production of IL-4, a cytokine which favors IgE class-switching and production by B cells. Finally, the role of MCs in aneurysmal progression was further analysed in vivo in ApoE-/- mice subjected to angiotensin II infusion aneurysm model, through MC-specific depletion after the establishment of dissecting aneurysms. MC-specific depletion improved intramural haematoma healing and reduced aneurysmal progression. CONCLUSIONS: Our data suggest that MC located close to aortic wall fissures are activated by adventitial TLO B cell-produced IgEs and participate to their own activation by providing support for further IgE synthesis through IL-4 production. By preventing prompt repair of aortic subclinical fissures, such a runaway MC activation loop could precipitate aneurysmal progression, suggesting that MC-targeting treatments may represent an interesting adjunctive therapy for reducing AAA progression.

A Novel Role of Semaphorin 3C in Modulating Systemic and Renal Hemodynamics.

BACKGROUND: Alterations of renal hemodynamics play an essential role in renal homeostasis and kidney diseases. Recent data indicated that semaphorin 3C (SEMA3C), a secreted glycoprotein involved in vessel development, can modulate renal vascular permeability in acute kidney injury, but whether and how it might impact systemic and renal hemodynamics is unknown. OBJECTIVES: The objective of the study was to explore the effect of SEMA3C on systemic and renal hemodynamics. METHODS: SEMA3C recombinant protein was administered intravenously in two-month-old wild-type mice, and the variations of mean arterial pressure, heart rate, renal blood flow, and renal vascular resistance were measured and analyzed. RESULTS: Acute administration of SEMA3C induced (i) systemic hemodynamic changes, including mean arterial pressure decrease and heart rate augmentation; (ii) renal hemodynamic changes, including reduced vascular resistance and elevated renal blood flow. Continuous perfusion of SEMA3C had no significant effect on systemic or renal hemodynamics. CONCLUSION: SEMA3C is a potent vasodilator affecting both systemic and renal hemodynamics in mice.

Heterozygous expression of Cre recombinase in podocytes has no impact on the anti-glomerular basement membrane glomerulonephritis model in C57BL/6J mice.

A recent article described a thickening of the glomerular basement membrane (GBM) along with changes in the expression of key components of the extracellular matrix in 6-month-old NPHS2-Cre transgenic mice, which express the Cre recombinase specifically in podocytes. This transgenic line has been widely used to characterize the implication of candidate genes in glomerular diseases in younger mice. Using a different mouse strain (C57BL/6J) than the previous report (129S6/SvEvTac), we sought to characterize 3- and 6-month-old NPHS2-Cre+/- mice in control and pathological conditions. At baseline, there was no difference in renal function and histology between control and NPHS2-Cre+/- mice. Notably, GBM thickness evaluated by transmission electron microscopy was similar between the two groups. We then induced an immune-mediated severe glomerular insult, the anti-glomerular basement membrane glomerulonephritis model (anti-GBM-GN) in 3-month-old control and NPHS2-Cre+/- mice. NPHS2-Cre+/- mice exhibited the same alterations in renal function and structure as control mice. In summary, our study strongly suggests that NPHS2-Cre+/- transgenic mice on a C57BL/6J background can be safely used for podocyte-specific gene inactivation in control conditions and in the anti-GBM-GN model.

Persistence of Intraluminal Thrombus Makes Saccular Aneurysm More Biologically Active than Fusiform in an Experimental Rat Model.

INTRODUCTION: Saccular aneurysms are thought to have a worse prognosis than fusiform aneurysms in humans, due to hemodynamic reasons. However, data comparing hemodynamic and biology in saccular and fusiform aneurysms are lacking. The main objective was to evaluate the impact of aneurysm morphology on intra-luminal thrombus (ILT) formation and activity. METHODS: Forty Lewis rats were ran-domly divided into 2 groups of 20: "saccular" (Group A) and "fusiform" (Group B) aneurysms. Decellularized thoracic aortas from guinea pigs were xenografted to create saccular or fusiform aneurysms. Final imaging evaluation of the aneurysms was carried out during the third week, by quantitative Doppler ultrasound and magnetic resonance imaging. Assays of myeloperoxidase (MPO), platelet factor 4 (PF4), advanced oxidation protein products (AOPPs) iron and matrix metallopeptidase-9 (MMP-9) were performed as biological criteria. RESULTS: Quantitatively, saccular aneurysms are characterized by a more thicker ILT, lower inflow velocities and more important relative backflow velocities as compared to fusiform aneurysms. Compared to fusiform, saccular aneurysms released significantly more MPO (p = 0.004), PF4 (p = 0.02), AOPPs (p < 0.002), iron (p < 0.0001) and MMP-9 (p < 0.04). CONCLUSION: Experimental saccular and fusiform aneurysms show differential specific hemodynamics, which seem to impact the histology and the biology of the ILT in each type of aneurysm.

Reconstituted High-density Lipoprotein Therapy Improves Survival in Mouse Models of Sepsis.

BACKGROUND: High-density lipoproteins exert pleiotropic effects including antiinflammatory, antiapoptotic, and lipopolysaccharide-neutralizing properties. The authors assessed the effects of reconstituted high-density lipoproteins (CSL-111) intravenous injection in different models of sepsis. METHODS: Ten-week-old C57BL/6 mice were subjected to sepsis by cecal ligation and puncture or intraperitoneal injection of Escherichia coli or Pseudomonas aeruginosa pneumonia. CSL-111 or saline solution was administrated 2 h after the sepsis. Primary outcome was survival. Secondary outcomes were plasma cell-free DNA and cytokine concentrations, histology, bacterial count, and biodistribution. RESULTS: Compared with saline, CSL-111 improved survival in cecal ligation and puncture and intraperitoneal models (13 of 16 [81%] survival rate vs. 6 of 16 [38%] in the cecal ligation and puncture model; P = 0.011; 4 of 10 [40%] vs. 0 of 10 [0%] in the intraperitoneal model; P = 0.011). Cell-free DNA concentration was lower in CSL-111 relative to saline groups (68 [24 to 123] pg/ml vs. 351 [333 to 683] pg/ml; P < 0.001). Mice injected with CSL-111 presented a decreased bacterial count at 24 h after the cecal ligation and puncture model both in plasma (200 [28 to 2,302] vs. 2,500 [953 to 3,636] colony-forming unit/ml; P = 0.021) and in the liver (1,359 [360 to 1,648] vs. 1,808 [1,464 to 2,720] colony-forming unit/ml; P = 0.031). In the pneumonia model, fewer bacteria accumulated in liver and lung of the CSL-111 group. CSL-111-injected mice had also less lung inflammation versus saline mice (CD68+ to total cells ratio: saline, 0.24 [0.22 to 0.27]; CSL-111, 0.07 [0.01 to 0.09]; P < 0.01). In all models, no difference was found for cytokine concentration. Indium bacterial labeling underlined a potential hepatic bacterial clearance possibly promoted by high-density lipoprotein uptake. CONCLUSIONS: CSL-111 infusion improved survival in different experimental mouse models of sepsis. It reduced inflammation in both plasma and organs and decreased bacterial count. These results emphasized the key role for high-density lipoproteins in endothelial and organ protection, but also in lipopolysaccharide/bacteria clearance. This suggests an opportunity to explore the therapeutic potential of high-density lipoproteins in septic conditions.

Early Detection of Localized Immunity in Experimental Autoimmune Myocarditis Using [99mTc]Fucoidan SPECT.

PURPOSE: The aim of the study was to evaluate the ability of technetium-99m-fucoidan ([99mTc]fucoidan), a molecular imaging agent specific for selectins, in the assessment of early localized immunity in a rat model of experimental autoimmune myocarditis (EAM). PROCEDURES: EAM was induced in Lewis rats and troponin T; brain natriuretic peptide (BNP) and anti-myosin antibodies were measured in plasma. Separately, [99mTc]fucoidan single-photon emission computed tomography (SPECT)/x-ray computed tomography (CT) was performed in the very early phase of myocarditis at 10, 15, and 21 days after immunization. Then, hearts were collected and used for autoradiography, well counting, histology, and flow cytometry analysis. RESULTS: The EAM acute phase is characterized by extensive myocardial necrosis, release of troponin and BNP, and pericardial effusion. [99mTc]Fucoidan uptake was significantly increased in EAM compared with controls starting from D15. There was a close relationship between uptake of the tracer and myocardial content in CD45+, CD8+, CD11b+, and CD31+ cells. CONCLUSIONS: [99mTc]Fucoidan SPECT/CT accurately diagnosed the autoimmune attack in the early steps of EAM and could be used to monitor disease evolution and therapy efficiency.

Astaxanthin Complexes to Attenuate Muscle Damage after In Vivo Femoral Ischemia-Reperfusion.

(1) Background: Reperfusion injury refers to the cell and tissue damage induced, when blood flow is restored after an ischemic period. While reperfusion reestablishes oxygen supply, it generates a high concentration of radicals, resulting in tissue dysfunction and damage. Here, we aimed to challenge and achieve the potential of a delivery system based on astaxanthin, a natural antioxidant, in attenuating the muscle damage in an animal model of femoral hind-limb ischemia and reperfusion. (2) Methods: The antioxidant capacity and non-toxicity of astaxanthin was validated before and after loading into a polysaccharide scaffold. The capacity of astaxanthin to compensate stress damages was also studied after ischemia induced by femoral artery clamping and followed by varied periods of reperfusion. (3) Results: Histological evaluation showed a positive labeling for CD68 and CD163 macrophage markers, indicating a remodeling process. In addition, higher levels of Nrf2 and NQO1 expression in the sham group compared to the antioxidant group could reflect a reduction of the oxidative damage after 15 days of reperfusion. Furthermore, non-significant differences were observed in non-heme iron deposition in both groups, reflecting a cell population susceptible to free radical damage. (4) Conclusions: Our results suggest that the in situ release of an antioxidant molecule could be effective in improving the antioxidant defenses of ischemia/reperfusion (I/R)-damaged muscles.

Neurologic Complications of Infective Endocarditis: A Joint Model for a Septic Thromboembolism and Inflammatory Small Vessel Disease.

OBJECTIVES: Embolic events from vegetations are commonly accepted as the main mechanism involved in neurologic complications of infective endocarditis. The pathophysiology may imply other phenomena, including vasculitis. We aimed to define the cerebral lesion spectrum in an infective endocarditis rat model. DESIGN: Experimental model of Staphylococcus aureus or Enterococcus faecalis infective endocarditis. Neurologic lesions observed in the infective endocarditis model were compared with three other conditions, namely bacteremia, nonbacterial thrombotic endocarditis, and healthy controls. SETTING: Research laboratory of a university hospital. SUBJECTS: Male Wistar rats. INTERVENTIONS: Brain MRI, neuropathology, immunohistochemistry for astrocyte and microglia, and bacterial studies on brain tissue were used to characterize neurologic lesions. MEASUREMENTS AND MAIN RESULTS: In the infective endocarditis group, MRI revealed at least one cerebral lesion in 12 of 23 rats (52%), including brain infarctions (n = 9/23, 39%) and cerebral microbleeds (n = 8/23, 35%). In the infective endocarditis group, neuropathology revealed brain infarctions (n = 12/23, 52%), microhemorrhages (n = 10/23, 44%), and inflammatory processes (i.e., cell infiltrates including abscesses, vasculitis, meningoencephalitis, and/or ependymitis; n = 11/23, 48%). In the bacteremia group, MRI studies were normal and neuropathology revealed only hemorrhages (n = 2/11, 18%). Neuropathologic patterns observed in the nonbacterial thrombotic endocarditis group were similar to those observed in the infective endocarditis group. Immunochemistry revealed higher microglial activation in the infective endocarditis group (n = 11/23, 48%), when compared with the bacteremia (n = 1/11, 9%; p = 0.03) and nonbacterial thrombotic endocarditis groups (n = 0/7, 0%; p = 0.02). CONCLUSIONS: This original model of infective endocarditis recapitulates the neurologic lesion spectrum observed in humans and suggests synergistic mechanisms involved, including thromboembolism and cerebral vasculitis, promoted by a systemic bacteremia-mediated inflammation.

An Experimental Study of Paclitaxel Embolisation During Drug Coated Balloon Angioplasty.

OBJECTIVE: Drug coated balloons (DCB) improve the patency of femoropopliteal angioplasty but their use in infrapopliteal lesions is debateable as paclitaxel (PTX) particle embolisation has been suspected in some trials. The aim of this study was to compare experimentally five DCBs in terms of distal embolism of PTX. METHODS: Twenty-five New Zealand rabbits were divided into five groups according to the DCB used: Lutonix (Bard), In.Pact (Medtronic), Passeo-18 Lux (Biotronik), Ranger (Boston Scientific), and Stellarex (Spectranetics) (n = 5 in each group). After ligation of the right common iliac artery, a 4 × 40 mm DCB was inflated in the infrarenal aorta for 180 seconds. Rabbits were euthanised two hours after inflation of the DCB. The infrarenal aorta, a blood sample and three left hind leg muscles (tensor fasciae latae [TFL], vastus lateralis [VL], and tibialis anterior [TA] muscles) were harvested for blind measurement of PTX concentrations and histological analysis (PTX emboli count). RESULTS: In the TA muscle (the most distal), concentrations of PTX were significantly lower for the Ranger (0.067 ng/mg) than for the Lutonix (0.342 ng/mg; p = .008), In.Pact (0.370 ng/mg; p = .012), and Passeo-18-Lux (0.160 ng/mg; p = .021) DCBs. Similarly, concentrations of PTX were significantly lower for the Passeo-18-Lux than for the In.Pact (p = .028). Concentrations of PTX were not significantly different between DCBs in the TFL and VL muscles. Concentrations of PTX were found to be significantly higher in the plasma and lower in the aorta and on the DCBs after use of Lutonix compared with the four other DCBs. Histological analysis revealed evidence of embolised PTX crystals in small arterioles of all muscle tissue samples without any significant difference between the DCBs. CONCLUSIONS: This study suggests some differences regarding distal embolisation profiles between the five assessed DCBs. Although clinical implications remain to be demonstrated, the present results may have implications when choosing a DCB, especially in a critical limb ischaemia setting.

Evaluation of some prostaglandins modulators on rat corpus cavernosum in-vitro: Is relaxation negatively affected by COX-inhibitors?

INTRODUCTION: Prostaglandins (PGs) play an important role in corpus cavernosum relaxation, as evidenced by alprostadil being used as a drug for erectile dysfunction. Reports about the effect of cyclooxygenase (COX) inhibitors on erectile function are highly contradictory. AIM: To compare the potential effects of some COX inhibitors with varying COX-1/COX-2 selectivities (indomethacin, ketoprofen and diclofenac) with that of the selective COX-2 inhibitor (DFU) on corpus cavernosal tone in-vitro. The role played by PGE1, PGI2-analogue and PGE4 receptor (EP4)-agonist in controlling corpus cavernosum function and the modulation of their action by sildenafil is also studied. METHODS: Organ bath experiments were performed using isolated rat corpus cavernosum. Direct relaxations and changes to electric field stimulation (EFS, 2-16 Hz, 60 V, 0.8 ms, 10 s train)-induced relaxation by the effect of the selected drugs were studied. Strips were precontracted using phenylephrine (PE, 10-5 M). Results are expressed as mean ± SEM of 5-9 rats. RESULTS: Alprostadil, iloprost and L902688 (selective EP4 agonist) induced direct relaxation where L902688 showed greater relaxant effect. Sildenafil potentiated the Emax of alprostadil and iloprost but not L902688. EFS and acetylcholine (ACh)-induced relaxations were significantly potentiated in presence of indomethacin, ketoprofen and diclofenac (20, 100 µM) but not in presence of selective COX-2 inhibitor (DFU, 1 µM). GR32191B (Thromboxane A2 receptor antagonist, 10-6 M) significantly reduced the potentiatory effect of indomethacin. Only diclofenac succeeded to potentiate sodium nitroprusside (SNP)-induced relaxation. CONCLUSIONS: EP4 receptors may play an important nitric oxide (NO)/cGMP-independent role in corpus cavernosal relaxation. Nonselective COX inhibitors seem of no harm concerning cavernosal tissue relaxation, possibly because they inhibit the synthesis of the highly contracting mediator thromboxane A2.

In vitro and in vivo evaluation of a dextran-graft-polybutylmethacrylate copolymer coated on CoCr metallic stent.

Introduction: The major complications of stent implantation are restenosis and late stent thrombosis. PBMA polymers are used for stent coating because of their mechanical properties. We previously synthesized and characterized Dextrangraft-polybutylmethacrylate copolymer (Dex-PBMA) as a potential stent coating. In this study, we evaluated the haemocompatibility and biocompatibility properties of Dex-PBMA in vitro and in vivo. Methods: Here, we investigated: (1) the effectiveness of polymer coating under physiological conditions and its ability to release Tacrolimus®, (2) the capacity of Dex-PBMA to inhibit Staphylococcus aureus adhesion, (3) the thrombin generation and the human platelet adhesion in static and dynamic conditions, (4) the biocompatibility properties in vitro on human endothelial colony forming cells ( ECFC) and on mesenchymal stem cells (MSC) and in vivo in rat models, and (5) we implanted Dex-PBMA and Dex-PBMATAC coated stents in neointimal hyperplasia restenosis rabbit model. Results: Dex-PBMA coating efficiently prevented bacterial adhesion and release Tacrolimus®. Dex-PBMA exhibit haemocompatibility properties under flow and ECFC and MSC compatibility. In vivo, no pathological foreign body reaction was observed neither after intramuscular nor intravascular aortic implantation. After Dex-PBMA and Dex-PBMATAC coated stents 30 days implantation in a restenosis rabbit model, an endothelial cell coverage was observed and the lumen patency was preserved. Conclusion: Based on our findings, Dex-PBMA exhibited vascular compatibility and can potentially be used as a coating for metallic coronary stents.

Peptide binding to cleaved CD31 dampens ischemia/reperfusion-induced intestinal injury.

BACKGROUND: CD31 is a key transmembrane neutrophil immunoregulatory receptor. Mesenteric ischemia/reperfusion-induced neutrophil activation leads to a massive cleavage and shedding of the most extracellular domains of CD31 into plasma, enhancing the deleterious effect of neutrophil activation. We have evaluated the preventive therapeutic potential of an engineered synthetic octapeptide (P8RI), which restores the inhibitory intracellular signaling of cleaved CD31, in an experimental model of acute mesenteric ischemia/reperfusion. METHODS: In a randomized, controlled, and experimenter-blinded preclinical study, mesenteric ischemia/reperfusion (I/R) was induced in Wistar rats by superior mesenteric artery occlusion for 30 min followed by 4 h of reperfusion. Three groups of rats were compared: I/R + saline perfusion (I/R controls group, n = 7), I/R + preventive P8RI perfusion (P8RI group, n = 7), and sham-operated rats + saline perfusion (sham group, n = 7). RESULTS: Compared with I/R controls, P8RI perfusion significantly decreased intestinal ischemia/reperfusion injury (Chiu's score, P = 0.01; epithelial area, P = 0.001) and bacterial translocation (plasma Escherichia coli DNA, P = 0.04) and could limit intestinal bleeding (P = 0.09). P8RI decreased neutrophil activation assessed by matrix metalloproteinase-9 release in plasma (P < 0.001) and in the intestinal wall, albeit without statistical significance (P = 0.06 and P = 0.058 for myeloperoxydase). Inhibition of CD31 cleavage from neutrophils could play a major role in the protective effects of P8RI (P < 0.0001). CONCLUSIONS: Preventive administration of P8RI, a CD31-agonist peptide, could decrease I/R-induced intestinal injury by potentially limiting neutrophil activation.

Downstream Microvascular Thrombosis in Cortical Venules Is an Early Response to Proximal Cerebral Arterial Occlusion.

BACKGROUND: Previous experimental studies have shown that downstream microvascular thromboinflammation is involved in brain damage from acute ischemic stroke. Using intravital microscopy, we investigated and characterized the sequence of downstream microvascular thromboinflammation in an ischemia/reperfusion acute ischemic stroke model. METHODS AND RESULTS: Rats underwent transient monofilament middle cerebral artery (MCA) occlusion. Cerebral microcirculation in the MCA territory was exposed through a craniotomy and analyzed using real-time intravital imaging coupled with laser Doppler interferometry. Leukocytes, platelets, fibrinogen, and blood-brain barrier permeability were analyzed by intravenous injection of fluorescent antibodies and bovine serum albumin. MCA occlusion induced a sudden and profound drop in downstream microvascular blood flow associated with leukocyte margination in the venous compartment. Leukocyte margination fostered fibrinogen deposition and thrombosis in postcapillary venules. Either in venules or arterioles, blood flow was not fully restored after MCA recanalization. Furthermore, venular thrombi persisted despite MCA recanalization, and leukocyte extravasation continued to develop in venules in association with blood-brain barrier disruption. Finally, microhemorrhages were occasionally observed, colocalizing with thrombosed venules characterized by marked leukocyte margination. CONCLUSIONS: We showed that microvascular thrombosis in transient monofilament MCA occlusion and blood-brain barrier disruption are initiated immediately after occlusion and are propagated through the venous compartment in close association with marginating leukocytes. MCA occlusion-induced downstream microvascular thromboinflammation response was responsible for incomplete reperfusion after MCA recanalization and delayed microhemorrhages.

Exploring antibody-dependent adaptive immunity against aortic extracellular matrix components in experimental aortic aneurysms.

BACKGROUND: Recent evidence suggests that adaptive immunity develops during abdominal aortic aneurysm evolution. Uncertainties remain about the antigens implicated and their role in inducing rupture. Because antigens from the extracellular matrix (ECM) have been suspected, the aim of this experimental study was to characterize the role of adaptive immunity directed against antigens from the aortic ECM. METHODS: In a first step, an experimental model of abdominal aortic aneurysm rupture based on adaptive immunity against the ECM was developed and characterized. Forty 4-week-old male Lewis rats were divided into two groups. In the ECM group (n = 20), rats were presensitized against the guinea pig aortic ECM before implantation of a decellularized aortic xenograft (DAX). In the control group (n = 20), rats were not presensitized before DAX implantation. In each group, half the rats were sacrificed at day 3 to analyze early mechanisms involved after DAX implantation. In a second step, we aimed to assess which ECM component was most efficient in inducing rupture. For this purpose, the nonfibrillar and fibrillar ECM components were sequentially extracted from the guinea pig aortic wall. Forty Lewis rats were then divided into four groups. Each group was presensitized against one ECM component (structural glycoproteins and proteoglycans, collagen, elastin alone, and elastin-associated glycoproteins) before DAX implantation. Apart from those that experienced rupture, rats were sacrificed at day 21. Xenografts were harvested for histologic, immunofluorescence, and conditioned medium analyses. RESULTS: In total, early aortic rupture occurred in 80% of the ECM group vs 0% of the control group (P < .001). In the ECM group, major circumferential immunoglobulin deposits were observed in combination with the C3 complement fraction, without cell infiltration. Conditioned medium analysis revealed that matrix metalloproteinase 9 and myeloperoxidase levels and elastase activities were significantly increased in this group. Immunofluorescence analysis demonstrated that myeloperoxidase co-localized with tissue-free DNA and histone H4, highlighting local neutrophil activation and formation of neutrophil extracellular traps. Following differential presensitization, it appeared that rats presensitized against structural glycoproteins and proteoglycans were significantly more susceptible to rupture after DAX implantation. CONCLUSIONS: Stimulating adaptive immunity against the aortic ECM, especially structural glycoproteins and proteoglycans, triggers rupture after DAX implantation. Further studies are needed to assess the precise proteins involved.

Efficacy of subcutaneous injection of platelet-rich plasma in alopecia: A clinical and histological pilot study on a rat model with a six-month long-term follow-up experience.

PURPOSE: To assess the potential of platelet-rich plasma (PRP) subcutaneous injection of to treat alopecia and to evaluate local toxicity. MATERIALS-METHODS: Twelve Hairless rats were used. At D0, we performed systematic clinical examination and divided the rat back into four quadrants (Q). We initiated subcutaneous injection using either PRP in PRPQ+, platelet-poor plasma (PPP) in PPPQ+, physiological serum (PS) in PSQ+, or no treatment (Q4). At D7, D14, D21, and D28 but also second month (M2), M3, M4, M5, rats had exactly the same injection procedure. Follow-up with PRP efficacy and toxicity at D28 and M6 using clinical and histological evaluation was performed. RESULTS: Hair density was significantly improved at D28 and at M6 for PRPQ+ vs PSQ+ (respectively, P = .0156 and P = .0313), PPPQ+ (respectively, P = .042 and P = .046). Significant histological improvement was observed between D28 and M6, for PRPQ+ vs PPPQ+ and PSQ+ for vessels (respectively, P = .0160 and P = .021), collagen (respectively, P = .0036 and P = .032), and epithelium (respectively, P = .0138 and P = .022) with no local toxicity. CONCLUSION: Our study suggests that subcutaneous PRP injections using controlled concentration of platelets and leukocytes improve hair growth.

Erythrocyte Efferocytosis by the Arterial Wall Promotes Oxidation in Early-Stage Atheroma in Humans.

BACKGROUND: Since red blood cells (RBCs) are the predominant cellular blood component interacting with the arterial wall, we explored the role of RBCs efferocytosis by vascular smooth muscle cells (vSMCs) in the initiation of human atheroma. METHODS AND RESULTS: The comparison of human healthy aortas with aortic fatty streaks or fibroatheromas revealed that RBC angiophagy is implicated from the earliest stages of atherogenesis, as documented by the concomitant detection of redox-active iron, hemoglobin, glycophorin A, and ceroids. RBCs infiltration in the arterial wall was associated with local lipid and protein oxidation, as well as vascular response (expression of heme oxygenase-1 and of genes related to iron metabolism as well as those encoding for phagocytosis). These effects were recapitulated in vitro when vSMCs were co-cultured with phosphatidyl-exposing senescent (s) RBCs but not with fresh RBCs. VSMCs engulfing sRBC increased their intracellular iron content, accumulated hemoglobin, lipids, and activated their phagolysosomes. Strikingly, injections of sRBCs into rats promoted iron accumulation in the aortic wall. In rabbits, hypercholesterolemia increased circulating senescent RBCs and induced the subendothelial accumulation of iron-rich phagocytic foam cells. RBCs bring cholesterol and iron/heme into the vascular wall and interact with vSMCs that phagocytize them. CONCLUSION: This study presents a previously unforeseen mechanism of plaque formation that implicates intimal RBC infiltration as one of the initial triggers for foam cell formation and intimal oxidation. Pathogenic effects exerted by several metabolic and hemodynamic factors may rely on their effect on RBC biology, thereby impacting how RBCs interact with the vascular wall.

Exacerbation of Thromboinflammation by Hyperglycemia Precipitates Cerebral Infarct Growth and Hemorrhagic Transformation.

BACKGROUND AND PURPOSE: Admission hyperglycemia is associated with a poor outcome in acute ischemic stroke. How hyperglycemia impacts the pathophysiology of acute ischemic stroke remains largely unknown. We investigated how preexisting hyperglycemia increases ischemia/reperfusion cerebral injury. METHODS: Normoglycemic and streptozotocin-treated hyperglycemic rats were subjected to transient middle cerebral artery occlusion. Infarct growth and brain perfusion were assessed by magnetic resonance imaging. Markers of platelet, coagulation, and neutrophil activation were measured in brain homogenates and plasma. Downstream microvascular thromboinflammation (DMT) was investigated by intravital microscopy. RESULTS: Hyperglycemic rats had an increased infarct volume with an increased blood-brain barrier disruption and hemorrhagic transformation rate compared with normoglycemic rats. Magnetic resonance imaging scans revealed that hyperglycemia enhanced and accelerated lesion growth and was associated with hemorrhagic transformation originating from territories that were still not completely reperfused at 1 hour after middle cerebral artery recanalization. Intravital microscopy and analysis of brain homogenates showed that DMT began immediately after middle cerebral artery occlusion and was exacerbated by hyperglycemia. Measurement of plasma serotonin and matrix metalloproteinase-9 indicated that platelets and neutrophils were preactivated in hyperglycemic rats. Neutrophils from hyperglycemic diabetic patients showed increased adhesion to endothelial cells as compared with neutrophils from normoglycemic donors in flow chamber experiments. CONCLUSIONS: We show that hyperglycemia primes the thromboinflammatory cascade, thus, amplifying middle cerebral artery occlusion-induced DMT. DMT exacerbation in hyperglycemic rats impaired reperfusion and precipitated neurovascular damage, blood-brain barrier disruption, and hemorrhagic transformation. Our results designate DMT as a possible target for reduction of the deleterious impact of hyperglycemia in acute ischemic stroke.