Altered Storage and Function of von Willebrand Factor in Human Cardiac Microvascular Endothelial Cells Isolated from Recipient Transplant Hearts.

The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel-Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMECD), or from nominally healthy donors (controls; HCMECC). Using fluorescence microscopy, WPBs in HCMECC (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMECD (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMECD revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMECD WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMECD were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.

Molecular analyses of human rabies virus associated with encephalitis in two children in Gabon.

Rabies is a zoonotic neurological life-threatening neglected tropical disease present worldwide, and Gabon is listed as an endemic country. However, despite strong clinical suspicion in humans and molecular confirmation of rabies virus (RABV) infections in dogs for several decades, no molecularly confirmed human case in Gabon has ever been reported. In this study, we describe two cases of human rabies and provide the first molecular diagnostic report on suspected human rabies cases in Gabon. Our results showed that the RABVs isolated from the patients are closely related to other RABV strains belonging to the African 1A subclade in the Cosmopolitan lineage isolated more than 20 years ago from Gabonese dog brains, suggesting that only this species circulates in the country. Because both patients had a history of dog bites a few weeks before symptom onset and the main causative agent of human rabies worldwide is dog-associated RABV, we conclude that dogs are likely to be the source of human infection in this study.

Leukocyte Adhesion Under Hemodynamic Flow Conditions.

Vascular endothelial cells (ECs) line the luminal side of all blood vessels and act as a selective barrier between blood and tissue. ECs are constantly exposed to biochemical and biomechanical stimuli from the blood and underlying tissue. Fluid shear stress acts in parallel to the vessel wall, resulting from friction of blood against EC. Despite the importance of flow on normal EC function, much of the information regarding EC function and dysfunction has been derived from cells harvested, grown, and studied in static culture.In order to study the effects of shear stress on EC function a number of in vitro models have been developed. This manuscript provides methodology for use of a system which enables recirculation of leukocytes and cell culture medium over the endothelium for a period of several minutes to days and enables investigation of the effects of prolonged leukocyte coculture on both the endothelial and leukocyte populations.

Blood transfusion, serum ferritin, and iron in hemodialysis patients in Africa.

Background and Objectives. There is no data analyzing the outcome of blood transfusions and oral iron therapy in patients with kidneys failure in sub-Saharan Africa. The present study aimed to fill that gap and assess the value of ferritin in the diagnosis of iron overload and deficiency. Design. From January to February 2012, we prospectively studied 85 hemodialysis patients (78% of males and 22% of females aged 20 to 79 years) attending the Gabonese National Hemodialysis Centre. Results. Correlation studies showed (a) a strong positive linear relationship between the number of blood transfusions and high serum ferritin in hemodialysis patient (Spearman r : 0.74; P value: 0.0001); (b) a weak association between the number of blood transfusions and serum iron concentrations (Spearman r : 0.32; P value: 0.04); (c) a weak association between serum ferritin and serum iron (Spearman r : 0.32; P value: 0.003). Also, the strength of agreement beyond chance between the levels of ferritin and iron in the serum was poor (κ = 0.14). The prevalence of iron overload was 10.6%, whereas the prevalence of iron deficiency was 2.3%, comparing (1) patients with a maximum of one transfusion not on iron therapy; (2) patients with a maximum of one transfusion on iron therapy; (3) polytransfused patients not on iron therapy; and (4) polytransfused patients on oral iron therapy. The "Kruskal-Wallis test" showed that ferritin levels varied significantly between the groups (P value: 0.0001). Conclusion. Serum ferritin is not reliable as a marker of iron overload. For patients undergoing regular transfusion we recommend routine serum ferritin measurement and yearly measurement of LIC.

Role of anti-vimentin antibodies in renal transplantation.

BACKGROUND: The role of non-HLA antibodies in rejection is not clear. We investigate whether antibodies to vimentin are made after renal transplantation and if production is associated with interstitial fibrosis and tubular atrophy (IFTA). METHODS: In this retrospective study, sera from 70 recipients of renal allografts (40 controls, 30 IFTA) were studied. The biopsy diagnosis of interstitial fibrosis and tubular atrophy (IFTA) was based on random, cause-indicating biopsies. Sera were collected pretransplant and at 3 monthly intervals up to 5 years posttransplant or diagnosis of IFTA and assayed by ELISA for IgM and IgG anti-vimentin antibodies (AVA) and HLA antibodies. RESULTS: Mean titers of IgM AVA were higher at every year after transplantation compared with pretransplant for both IFTA and controls groups (P<0.001). There was no difference in the mean level of IgM AVA achieved by IFTA and control groups. The mean pretransplant levels of IgG AVA in the IFTA and control group were 18.2±11.7 and 11.0±8.1, respectively (P=0.001). There was a significant increase between the pretransplant mean levels of IgG AVA and the levels at years 1 to 4 in the IFTA group (years 1-3, P<0.0001, year 4 P=0.003) but not in the controls. There was no significant difference between the numbers of IFTA or control patients achieving a positive value (mean+2SD of pretransplant antibody titers) of IgM AVA (50% versus 37.5%, respectively) or IgG AVA (26.6% versus 12.5%, respectively). There was no association between production of HLA and AVA antibodies. CONCLUSION: Posttransplant production of IgM AVA is not associated with IFTA. The production of IgG AVA by a minority of IFTA patients suggests that in some individuals, IgG AVA may be involved in the pathology of IFTA.

Pre-transplant donor HLA-specific antibodies: characteristics causing detrimental effects on survival after lung transplantation.

BACKGROUND: The impact of Luminex-detected HLA antibodies on outcomes after lung transplantation is unclear. Herein we have undertaken a retrospective study of pre-transplant sera from 425 lung transplants performed between 1991 and 2003. METHODS: Pre-transplant sera, originally screened by complement-dependent cytotoxicity (CDC) assays, were retrospectively tested for the presence of HLA-specific antibodies using HLA-coated Luminex beads and C4d deposition on Luminex beads. The results were correlated with graft survival at 1 year. RESULTS: Twenty-seven patients were retrospectively identified as having been transplanted against donor-specific HLA antibodies (DSA) and 36 patients against non-donor-specific HLA antibodies (NDSA). DSA-positive patients had 1-year survival of 51.9% compared with 77.8% for NDSA and 71.8% for antibody-negative patients (p = 0.029). One-year survival of patients with complement-fixing DSA was 12.5% compared with 62.5% for non-complement-fixing DSA, 75.8% for non-complement-fixing NDSA and 71.8% for antibody-negative patients (p < 0.0001). DSA-positive patients with mean fluorescence intensity (MFI) >5,000 had 1-year survival of 33.3% compared with 71.4% for MFI 2,000 to 5000 and 62.5% for MFI <2,000 (p = 0.0046). Multivariable analysis revealed DSA to be an independent predictor of poor patient survival within 1 year (p = 0.0010, hazard ratio [HR] = 3.569) as well as complement-fixing DSA (p < 0.0001, HR = 11.083) and DSA with MFI >5,000 (p = 0.0001, HR = 5.512). CONCLUSIONS: Pre-formed DSA, particularly complement-fixing DSA, and high MFI are associated with poor survival within the first year after lung transplantation. Risk stratification according to complement fixation or MFI levels may allow for increased transplantation in sensitized patients.

Heat shock protein-27 delays acute rejection after cardiac transplantation: an experimental model.

BACKGROUND: Rejection is the major obstacle to survival after cardiac transplantation. We investigated whether overexpression of heat shock protein (Hsp)-27 in mouse hearts protects against acute rejection and the mechanisms of such protection. METHODS: Hearts from B10.A mice overexpressing human Hsp-27 (Hsp-27tg), or Hsp-27-negative hearts from littermate controls (LCs) were transplanted into allogeneic C57BL/6 mice. The immune response to B10.A hearts was investigated using quantitative polymerase chain reaction for CD3+, CD4+, CD8+ T cells, and CD14+ monocytes and cytokines (interferon-γ, interleukin [IL]-2, tumor necrosis factor-α, IL-1ß, IL-4, IL-5, IL-10, transforming growth factor-ß) in allografts at days 2, 5, and 12 after transplantation. The effect of Hsp-27 on ischemia-induced caspase activation and immune activation was investigated. RESULTS: Survival of Hsp-27tg hearts (35±10.37 days, n=10) was significantly prolonged compared with LCs (13.6±3.06 days, n=10, P=0.0004). Hsp-27tg hearts expressed significantly more messenger RNA (mRNA) markers of CD14+ monocytes at day 2 and less mRNA markers of CD3+ and CD8+T cells at day 5 compared with LCs. There was more IL-4 mRNA in Hsp-27tg hearts at day 2 and less interferon-γ mRNA at day 5 compared with LCs. Heat shock protein-27tg hearts subjected to ischemia or to 24 hr ischemia-reperfusion injury demonstrated significantly less apoptosis and activation of caspases 3, 9, and 1 than LCs. T cells removed from C57BL/6 recipients of Hsp-27tg hearts produced a vigorous memory response to B10.A antigens, suggesting immune activation was not inhibited by Hsp-27. CONCLUSION: Heat shock protein-27 delays allograft rejection, by inhibiting tissue damage, through probably an antiapoptotic pathway. It may also promote an anti-inflammatory subset of monocytes.

Role of anti-vimentin antibodies in allograft rejection.

Production of anti-vimentin antibodies (AVA) after solid organ transplantation are common. Although classically thought to be expressed mainly within the cytosol, recent evidence demonstrates that extracellular or cell surface expression of vimentin is not unusual. This review examines the evidence to assess whether AVA contribute to allograft pathology. Clinical studies suggest that AVA are associated with cardiac allograft vasculopathy in heart transplant recipients. Studies in non-human primates confirm that production of AVA after renal and heart transplantation are not inhibited by Cyclosporine. Experimental studies have demonstrated that mice pre-immunised with vimentin undergo accelerated acute rejection and vascular intimal occlusion of cardiac allografts. Adoptive transfer of hyperimmune sera containing AVA into B-cell-knock-out mice caused accelerated rejection of allografted hearts, this is clear evidence that antibodies to vimentin accelerate rejection. AVA act in concert with the alloimmune response and AVA do not damage syngeneic or native heart allografts. Confocal microscopy of allografted organs in vimentin immunised mice shows extensive expression of vimentin on endothelial cells, apoptotic leukocytes and platelet/leukocyte conjugates, co-localising with C4d. One explanation for the ability of AVA to accelerate rejection would be fixation of complement within the graft and subsequent pro-inflammatory effects; there may also be interactions with platelets within the vasculature.

Solid phase assay measuring C4d deposition to determine complement fixation by HLA-specific antibodies.

The definition of HLA-specific antibodies in solid organ transplant patients is a necessary tool for recipient selection prior to transplantation and monitoring for rejection post transplant. Solid phase assays can detect both complement fixing and non-complement fixing HLA-specific antibodies. Here we describe a method for determining the presence of complement fixing HLA-specific antibodies using a sensitive solid phase assay.

Antibody alone is not a stimulator of exocytosis of Weibel-Palade bodies from human endothelial cells.

BACKGROUND: The mechanisms of antibody-mediated damage to allografts are not well understood. We have examined the effect of antibodies to human leukocyte antigens on secretion of von Willebrand factor (vWF) from endothelial cells (ECs). METHODS: The effect of monoclonal antibodies (W6/32, L2, and L243), in the presence and absence of sublytic concentrations of complement, on the release of vWF from Weibel-Palade bodies (WPBs) in human umbilical vein ECs (HUVECs), human aortic ECs (HAECs), and human heart microvascular ECs (HHMECs) was investigated using biochemical and live-cell imaging. Fura-2-loaded ECs expressing the WPB marker proregion-enhanced green fluorescence protein were imaged simultaneously for intracellular Ca(2+) changes ([Ca(2+)](i)) and WPB exocytosis. RESULTS: Stimulation of ECs with 1- or 10-µg/mL W6/32, L2, or L243 did not evoke significant vWF release above control IgG. In live-cell imaging studies, exposure of proregion-enhanced green fluorescence protein-expressing HAECs to physiologic saline, 10-µg/mL U9F4, or W6/32 alone for 5 to 10 min induced irregular (Ca(2+))(i)\ spiking but no WPB exocytosis. Histamine-evoked WPB exocytosis was not changed by preexposure of HAECs to physiologic saline, U9F4, or W6/32. Stimulation of HUVECs with sublytic complement concentrations evoked WPB exocytosis; however, the addition of W6/32 did not change the amount of vWF released. CONCLUSION: Antibodies to human leukocyte antigen class I or II do not elicit significant WPB exocytosis or vWF secretion from ECs in the absence of exogenous complement.

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis.

Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis.

Genetic or pharmaceutical blockade of phosphoinositide 3-kinase p110δ prevents chronic rejection of heart allografts.

Chronic rejection is the major cause of long-term heart allograft failure, characterized by tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signaling, regulating both T cell activation and migration of primed T cells to non-lymphoid antigen-rich tissue. We investigated the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model in which HY-mismatched male hearts were transplanted into female recipients. We show that suppression of p110δ activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment by impairing the localization of antigen-specific T cells to the grafts, while not inducing specific T cell tolerance. p110δ pharmacologic inactivation is effective when initiated after transplantation. Targeting p110δ activity might be a viable strategy for the treatment of heart chronic rejection in humans.

Late antibody-mediated rejection after heart transplantation following the development of de novo donor-specific human leukocyte antigen antibody.

BACKGROUND: Antibody-mediated rejection (AMR) is an important problem after heart transplantation. Most cases seem to occur in sensitized recipients with preformed donor-specific human leukocyte antigen antibody (DSA) early after transplantation. Few data exist on AMR in patients who form de novo DSA. We describe the clinical features and treatment outcome for late AMR secondary to de novo DSA. METHODS: This was a retrospective, observational cohort study. All heart transplant patients treated for symptomatic AMR secondary to de novo DSA between November 2005 and August 2011. RESULTS: Fifteen patients were treated for AMR giving an incidence of 3.1 cases per 1000 person years and a prevalence of 1.4%. All had evidence of heart failure on presentation and de novo DSA at diagnosis. There was a spectrum of histologic and immunohistochemical findings. Despite treatment with immunepheresis, intravenous immunoglobulin, and rituximab, and in some cases total lymph node irradiation (n=3) and bortezomib (n=2), clinical outcomes were poor. DSA antibody levels, measured using Labscreen single antigen kits, were reduced by a mean of 76% with a median of 77% and a range of 35% to 99%, but were not eliminated. Forty-six percent had persistent cardiac allograft dysfunction. Mean and median survival was 1.3 and 0.8 years after diagnosis of AMR. Only 40% were alive at the end of the study period. CONCLUSION: Late cardiac AMR caused by de novo DSA was an uncommon but serious problem. Despite treatment consistent with current best practice, 46% of patients developed persistent cardiac dysfunction and their medium-term survival was poor.

Accommodation: does it apply to human leukocyte antigens?

The term "accommodation" was invoked to describe endothelial cell resistance to antibody-mediated rejection after ABO-incompatible kidney or experimental xenograft transplantation. Currently, there is much interest in how to achieve successful human leukocyte antigen (HLA)-incompatible allograft transplantation in HLA-sensitized patients and how to treat antibody-mediated rejection after ABO-compatible HLA-incompatible allotransplantation. The term "accommodation" is often used interchangeably to describe patients who have donor-specific ABO or HLA alloantibodies in the absence of damage to their allograft. Here, we suggest that there are important differences between the immune responses to protein versus carbohydrate antigens and that graft HLA molecules may respond differently to antibodies (and antibody isotypes) than ABO antigens. Neither the mechanisms nor a phenotype of accommodation have been defined fully. Further research is needed to define mechanisms of both resistance and susceptibility to antibody-mediated injury and to predict under which circumstances allograft accommodation may occur.

HLA and MICA: targets of antibody-mediated rejection in heart transplantation.

BACKGROUND: The goal of this study was to determine whether antidonor antibodies directed against human leukocyte antigen (HLA) or endothelial cells (ECs) expressed antigens, including major histocompatibility complex class I chain-related antigens A (MICA) are associated with the diagnosis of antibody-mediated rejection (AMR) in heart transplant recipients. METHODS: We studied posttransplant antidonor HLA antibodies in 168 heart allograft recipients transplanted from October 2001 to December 2005. Among them, there were 37 AMR+ patients and 131 age- and sex- matched AMR- controls. Sera were collected at the time of protocol biopsies and tested for the presence of HLA antibodies. Seventy-two of the 168 patients were genotyped for donor and recipient MICA alleles and were tested for the presence of anti-MICA antibodies. Thirty-one patients who never developed antibodies to HLA or MICA were further tested for anti-EC antibodies. RESULTS AND CONCLUSIONS: Of 37 AMR+ patients, 22 (60%) developed donor-specific antibodies (DSA) to HLA compared with 6 of 131(4%) AMR- patients (P<0.0001). Of the remaining 15 AMR+ patients, 5 had anti-HLA antibodies that were not donor specific and 10 did not show any HLA antibodies. In the subgroup of 72 patients, all 19 AMR+ patients had clearly demonstrable antibodies reactive with donor HLA, MICA or with nondonor-derived ECs, with 30% of them showed antibodies directed to non-HLA antigens. The incidence of transplant coronary artery disease was significantly higher in patients who had DSA to HLA and MICA compared with patients without DSA.

Report from a consensus conference on antibody-mediated rejection in heart transplantation.

BACKGROUND: The problem of AMR remains unsolved because standardized schemes for diagnosis and treatment remains contentious. Therefore, a consensus conference was organized to discuss the current status of antibody-mediated rejection (AMR) in heart transplantation. METHODS: The conference included 83 participants (transplant cardiologists, surgeons, immunologists and pathologists) representing 67 heart transplant centers from North America, Europe, and Asia who all participated in smaller break-out sessions to discuss the various topics of AMR and attempt to achieve consensus. RESULTS: A tentative pathology diagnosis of AMR was established, however, the pathologist felt that further discussion was needed prior to a formal recommendation for AMR diagnosis. One of the most important outcomes of this conference was that a clinical definition for AMR (cardiac dysfunction and/or circulating donor-specific antibody) was no longer believed to be required due to recent publications demonstrating that asymptomatic (no cardiac dysfunction) biopsy-proven AMR is associated with subsequent greater mortality and greater development of cardiac allograft vasculopathy. It was also noted that donor-specific antibody is not always detected during AMR episodes as the antibody may be adhered to the donor heart. Finally, recommendations were made for the timing for specific staining of endomyocardial biopsy specimens and the frequency by which circulating antibodies should be assessed. Recommendations for management and future clinical trials were also provided. CONCLUSIONS: The AMR Consensus Conference brought together clinicians, pathologists and immunologists to further the understanding of AMR. Progress was made toward a pathology AMR grading scale and consensus was accomplished regarding several clinical issues.

The indirect alloimmune response causes microvascular endothelial dysfunction-a possible role for alloantibody.

BACKGROUND: The causes of endothelial dysfunction after cardiac transplantation are unknown. Here, we have investigated whether the indirect alloimmune response mediates endothelial dysfunction in a major histocompatibility complex class I mismatch model. METHODS: PVG.RT1 rat hearts were transplanted into thymectomized CD8 T-cell-depleted allogeneic (PVG.R8) or syngeneic (PVG.RT1) recipients. Alloantibody was assessed at 2, 4, and 8 weeks. Cardiac allograft vasculopathy, the nature of the inflammatory infiltrate, and origin of endothelial cells were examined at 1, 2, 4, and 8 weeks. Endothelial function was assessed by Langendorff preparations at 1, 2, and 4 weeks. RESULTS: Recipients produced alloantibody and showed luminal occlusion at 1 (17.7%±8.0%), 2 (23.2%±4.9%), 4 (34.3%±5.0%), and 8 weeks (58.1%±1.8%) posttransplantation. The major inflammatory features of the allografts consisted of CD11b monocytes, CD4 T cells, and C4d deposition. At 1 week, the basal coronary flow and the vasodilator response to 5-hydroxytrytamine of syngeneic and allografted hearts were inhibited compared with normal hearts. At 4 weeks, the basal coronary flow of allografts was 54% lower than syngrafts (P<0.01), and 5- hydroxytrytamine and sodium nitroprusside did not evoke an increase in coronary flow in the allograft heart compared with syngeneic controls (P<0.01). Culture of aortic rings with antibody to major histocompatibility complex class I inhibited endothelium-dependent vasodilation to acetylcholine. CONCLUSION: Transient microvascular endothelial dysfunction occurred in syngeneic and allogeneic cardiac grafts after transplantation. Syngeneic but not allogeneic grafts recovered, suggesting the indirect immune response, consisting of CD4 T cells, monocytes, and antibody, mediates endothelial dysfunction. A possible role for alloantibody in endothelial dysfunction is discussed.

Protective effect of phosphorylated Hsp27 in coronary arteries through actin stabilization.

There is evidence for an inverse association between cellular expression of Hsp27 and vascular disease with carotid plaques, endarterectomy specimens, and cardiac biopsies investigated to date. Here we compare non-diseased coronary arteries from human heart transplant donors and patients with dilated cardiomyopathy (DCM) with no evidence of coronary artery disease, to coronary arteries from patients with ischemic heart disease (IHD) in order to determine abundance of phosphorylated Hsp27 (phospho-Hsp27) in plaque-free diseased vessels and elucidate how this protective effect is brought about through protein regulation. Western blotting identified phospho-Hsp27, phosphorylated on Ser82, Ser78, and Ser15, to be specifically decreased in IHD, but not DCM, compared to non-diseased vessels. Immunohistochemistry confirmed these results and revealed phospho-Hsp27 was located within both smooth muscle and endothelial cells. Disease-free coronary arteries and from patients with IHD were then subjected to 2-Dimensional Difference Gel Electrophoresis (2D-DIGE) analysis to detect proteins with altered abundance, which were subsequently identified by mass spectrometry. Hsp27 showed decreased abundance in ischemic vessels as expected. The expression of cytoskeletal proteins, namely vimentin was significantly reduced, while transgelin and tropomyosin showed significantly increased abundance in vessels with IHD. Immunohistochemistry studies suggested an increase in G-actin abundance to be present within IHD vessels. The results are consistent with the hypothesis that phospho-Hsp27 protects against vascular disease possibly by stabilizing the actin cytoskeleton within endothelial and/or smooth muscle cells.

Autoimmunity to vimentin potentiates graft vasculopathy in murine cardiac allografts.

BACKGROUND: There is increasing evidence for a role for autoimmunity in transplant rejection. It has previously been shown that autoantibodies to vimentin (Vim) accelerate acute rejection of murine cardiac allografts. We have investigated whether autoimmunity to Vim contributes to development of cardiac allograft vasculopathy (CAV). METHODS: Two well-established minor mismatch murine models of CAV were used, transplantation of 129/sv hearts into T-cell-depleted C57Bl/6 (B6) recipients and transplantation of FVB hearts into nonimmunosuppressed DBA/1 recipients. Recipients were immunized with recombinant mouse Vim in complete Freunds adjuvant, and controls received hen egg lysozyme 2 weeks before transplantation. T cell and antibody responses to Vim were assessed by ELISPOT and ELISA, respectively. CAV within transplanted hearts was assessed by quantitative morphometry of occluded vessels, presence of smooth muscle cells, deposition of C3d, and confocal microscopy. RESULTS: Allografts were harvested from B6 recipients at days 30 and 45 and from DBA/1 recipients at days 18 and 35. At all days, there was significantly more intimal occlusion of arteries of Vim -immunized mice than controls. There was significantly more smooth muscle cell alpha actin in vessels from Vim-immunized mice, and more C3d deposited in hearts from Vim-immunized mice. Confocal microscopy demonstrated colocalization of Vim with C3d on endothelial cells, leukocytes, and platelets in allogeneic but not syngeneic hearts. Serum from Vim-immunized mice, but not controls, caused platelet/leukocyte conjugation when added to mouse leukocytes. CONCLUSION: The autoimmune response to Vim accelerates CAV progression in these minor-mismatched models.