Cecilia Österholm

CTLA4Ig combined with anti-LFA-1 prolongs cardiac allograft survival indefinitely

CTLA4Ig and anti-LFA-1 are members of a new generation of immunomodulatory drugs which inhibit important signaling pathways in T cell activation. Both substances target molecules which have pivitol functions in the activation of CD4+ and CD8+ T cells and have been theorized to have an interdependent relationship. These drugs have been used independently in various treatment regimens and have shown great promise in prolonging the survival of allografts. In order to test whether these substances have synergistic or potentiating effects when combined, we performed mixed lymphocyte reactions, skin transplantation and vascularised heterotopic heart transplantation in the Balb/c (H-2(d)) to C3H/HeJ (H-2(k)) strain combination. When anti-LFA-1 and CTLA4Ig were combined at low doses, there was a substantial inhibition of lymphocyte proliferation. When each drug was used as a mono-therapy in skin graft recipients, there was no significant effect on median graft survival (anti-LFA-1, 15 days; CTLA4Ig, 16 days) when compared to untreated controls (13 days), whereas a combination of anti-LFA-1 and CTLA4Ig extended graft survival significantly to 32 days. Untreated vascularised heart grafts rejected at a median of 8 days, CTLA4Ig-treated mice rejected at a median time of 79 days and anti-LFA-1-treated mice rejected at 43 days (n = 9). When CTLA4Ig and anti-LFA-1 were combined, all animals had functioning heart grafts at 100 days after transplantation. Histological analysis of combined-therapy hearts showed no signs or only minor changes associated with chronic rejection. In conclusion, these results indicate a synergistic effect of combining anti-LFA-1 with CTLA4Ig in inhibiting lymphocyte proliferation and prolonging the survival of fully MHC-mismatched allografts.

Profiling of Atherosclerotic Lesions by Gene and Tissue Microarrays Reveals PCSK6 as a Novel Protease in Unstable Carotid Atherosclerosis

Objective—Carotid plaque instability is a major cause of ischemic stroke, but detailed knowledge about underlying molecular pathways is still lacking. Here, we evaluated large-scale transcriptomic and protein expression profiling in a biobank of carotid endarterectomies followed by characterization of identified candidates, as a platform for discovery of novel proteins differentially regulated in unstable carotid lesions. Approach and Results—Genes highly upregulated in symptomatic versus asymptomatic plaques were selected from Affymetrix microarray analyses (n=127 plaques), and tissue microarrays constructed from 34 lesions were assayed for 21 corresponding proteins by immunohistochemistry. Quantification of stainings demonstrated differential expression of CD36, CD137, and DOCK7 (P<0.05) in unstable versus stable lesions and the most significant upregulation of a proprotein convertase, PCSK6 (P<0.0001). Increased expression of PCSK6 in symptomatic lesions was verified by quantitative real-time polymerase chain reaction (n=233), and the protein was localized to smooth muscle &agr;-actin positive cells and extracellular matrix of the fibrous cap by immunohistochemistry. PCSK6 expression positively correlated to genes associated with inflammation, matrix degradation, and mitogens in microarrays. Stimulation of human carotid smooth muscle cells in vitro with cytokines caused rapid induction of PCSK6 mRNA. Conclusions—Using a combination of transcriptomic and tissue microarray profiling, we demonstrate a novel approach to identify proteins differentially expressed in unstable carotid atherosclerosis. The proprotein convertase PCSK6 was detected at increased levels in the fibrous cap of symptomatic carotid plaques, possibly associated with key processes in plaque rupture such as inflammation and extracellular matrix remodeling. Further studies are needed to clarify the role of PCSK6 in atherosclerosis.

Anti-lymphocyte function-associated antigen-1 monoclonal antibody inhibits CD40 ligand-independent immune responses and prevents chronic vasculopathy in CD40 ligand-deficient mice.

Background. Blockade of CD40 ligand (CD40L; CD154, gp39) is a potential treatment for autoimmune disease and allograft rejection. However, CD40L−/− mice are capable of mobilizing cellular immune responses to viral, parasitic, and intracellular bacterial infections as well as rejecting skin grafts with nearly the same efficiency as wild-type mice. CD40L-deficient mice (CD40L−/−) or wild-type mice treated with anti-CD40L develop chronic vasculopathy only 8 weeks after allogeneic heart transplantation. To overcome CD40L-independent immune responses, we used anti-lymphocyte function-associated antigen monoclonal antibody (LFA)-1, which has previously been shown to inhibit CD8+ immune responses. Methods. We conducted mixed lymphocyte reactions, cytotoxicity assays, skin transplantation, and vascularized heterotopic heart transplantation in wild-type B6 and CD40L-deficient mice in the presence and absence of anti-LFA-1 to study the effects of anti-LFA-1 in the absence of CD40L signaling. Results. Anti-LFA-1 inhibited proliferation of naïve CD40L−/− mixed leukocyte reactions and the lysis of donor targets by CD40L−/− cytotoxic T lymphocytes. Anti-LFA-1–treated CD40L−/− mice that received fully MHC-mismatched skin grafts showed significant prolongation of graft survival, with a median survival time of 55 days (mean 66 days) compared with 13 and 21 days in wild-type and CD40L−/− controls, respectively. CD40L−/− mice that received fully MHC-mismatched vascularized heart transplants treated with four doses of 200 &mgr;g of anti-LFA-1 at the time of transplantation did not develop any signs of chronic vasculopathy 150 days after transplantation. Conclusion. These results indicate that anti-LFA-1 can complement CD40L inhibition in the prevention of undesirable immune responses.

Increased expression of heparanase in symptomatic carotid atherosclerosis

OBJECTIVE Proliferation of smooth muscle cells (SMCs) can stabilize atherosclerotic lesions but the molecular mechanisms that regulate this process in humans are largely unknown. We have previously shown that heparan sulfate proteoglycans (HSPGs), such as perlecan, regulate SMC growth in animal models by modulating heparin-binding mitogens. Since perlecan is expressed at low levels in human atherosclerosis, we speculated that the effect of heparan sulfate (HS) in human disease was rather influenced by HS degradation and investigated the expression of heparanase (HPSE) in human carotid endarterectomies. METHODS AND RESULTS Gene expression analysis from 127 endarterectomies in the BiKE database revealed increased expression of HPSE in carotid plaques compared with normal arteries, and a further elevation in symptomatic lesions. Increased HPSE protein expression in symptomatic plaque tissue was verified by tissue microarrays. HPSE mRNA levels correlated positively with expression of inflammatory markers IL-18, RANTES and IL-1β, and also T-cell co-stimulatory molecules, such as B7.2, CD28, LFA-1 and 4-1BB. Previously reported single nucleotide polymorphisms within HPSE were associated with differential mRNA expression in plaques. Immunohistochemistry revealed that inflammatory cells were major producers of HPSE in plaque tissue. HPSE immunoreactivity was also observed in SMCs adjacent to the necrotic core and was co-localized to deposits of fibrin. CONCLUSIONS This study demonstrates increased expression of HPSE in human atherosclerosis associated with inflammation, coagulation and plaque instability. Since HS can regulate SMC proliferation and influence plaque stability, the findings suggest that HPSE degradation of HS take part in the regulation of SMC function in human atherosclerosis.

CTLA4ig induces long-term graft survival of allogeneic skin grafts and totally inhibits T-cell proliferation in LFA-1-deficient mice.

BACKGROUND It was recently shown that some strains of mice are capable of rejecting transplants independently of B7 and CD40L signaling and that this rejection is mediated by CD8(+) T cells. LFA-1 is known to be important for CD8(+) T cell activation and cytotoxicity. Therefore, blockade of LFA-1 could be important in overcoming costimulation blockade, CD8(+) T-cell-mediated, resistant rejection. The purpose of this study was to define the effect of combined blockade of the LFA-1 and B7 costimulation pathways on the alloimmune response in mice. METHODS Allogeneic skin transplantation was performed using BALB/c mice as donors and C57BL/6J wild-type or LFA-1-deficient (CD11a(-/-)) mice as recipients. CTLA4Ig or anti-LFA-1 was administered either as an induction or a prolonged therapy. Mixed lymphocyte reactions were conducted to study the effect of CTLA4Ig on T-cell proliferation in CD11a(-/-) mice. RESULTS AND CONCLUSIONS Administration of CTLA4Ig completely inhibits CD11a(-/-) T-cell proliferation in response to alloantigens and significantly improved skin allograft survival in CD11a(-/-) mice. Prolonged treatment of wild-type recipient mice with CTLA4Ig and anti-LFA-1 increased median survival time to 45.5 days compared with 16 days after induction therapy, but it was not sufficient to induce indefinite allograft survival in this model.

MicroRNA-210 Enhances Fibrous Cap Stability in Advanced Atherosclerotic Lesions

Rationale: In the search for markers and modulators of vascular disease, microRNAs (miRNAs) have emerged as potent therapeutic targets. Objective: To investigate miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. Methods and Results: Using patient material from the BiKE (Biobank of Karolinska Endarterectomies), we profiled miRNA expression in patients with stable versus unstable carotid plaque. A polymerase chain reaction–based miRNA array of plasma, sampled at the carotid lesion site, identified 8 deregulated miRNAs (miR-15b, miR-29c, miR-30c/d, miR-150, miR-191, miR-210, and miR-500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser capture microdissection and in situ hybridization revealed a distinct localization of miR-210 in fibrous caps. We confirmed that miR-210 directly targets the tumor suppressor gene APC (adenomatous polyposis coli), thereby affecting Wnt (Wingless-related integration site) signaling and regulating smooth muscle cell survival, as well as differentiation in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in 2 rodent models of vascular remodeling and plaque rupture. Modulating miR-210 in vitro and in vivo improved fibrous cap stability with implications for vascular disease. Conclusions: An unstable carotid plaque at risk of stroke is characterized by low expression of miR-210. miR-210 contributes to stabilizing carotid plaques through inhibition of APC, ensuring smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic vascular events.

Antenatal imatinib treatment reduces pulmonary vascular remodeling in a rat model of congenital diaphragmatic hernia

The pathophysiology of congenital diaphragmatic hernia (CDH) is constituted by pulmonary hypoplasia and pulmonary hypertension (PH). We previously reported successful treatment with imatinib of a patient with CDH. This study examines the effect of antenatal imatinib administration on the pulmonary vasculature in a rat model of CDH. Pregnant rats were given nitrofen to induce CDH. Controls were given olive oil. Half of the CDH fetuses and half of the controls were treated with imatinib antenatally E17-E21, rendering four groups: Control, Control+Imatinib, CDH, and CDH+Imatinib. Lung sections were obtained for morphometry and immunohistochemistry, and protein was purified for Western blot. Effects of nitrofen and imatinib on Ki-67, caspase-3, PDGF-B, and PDGF receptors were analyzed. Imatinib significantly reduced medial wall thickness in pulmonary arteries of rats with CDH. It also normalized lumen area and reduced the proportion of fully muscularized arteries. Imatinib also caused medial thinning in the control group. Cell proliferation was increased in CDH, and this proliferation was significantly reduced by imatinib. PDGF-B and PDGFR-β were upregulated in CDH, and imatinib treatment resulted in a downregulation. PDGFR-α remained unchanged in CDH but was significantly downregulated by imatinib. Antenatal imatinib treatment reduces development of medial wall thickness and restores lumen area in pulmonary arteries in nitrofen-induced CDH. The mechanism is reduced cell proliferation. Imatinib is an interesting candidate for antenatal therapy for PH in CDH, but potential side effects need to be investigated and more specific targeting of PDGF signaling is needed.

Anti-LFA-1 Improves Pig Islet Xenograft Function in Diabetic Mice When Long-Term Acceptance Is Induced by CTLA4Ig/Anti-CD40L.

Background. It has been previously demonstrated that addition of anti-LFA-1 to a combination of CTLA4Ig and anti-CD40L induces the permanent acceptance of dopaminergic fetal pig xenografts when transplanted into the brain of wild-type mice. The purpose of this study was to test whether this costimulation blockade also can induce acceptance of adult pig islets transplanted to C57BL/6 mice with streptozotocin-induced diabetes. Methods. Recipients were treated with CTLA4Ig/anti-CD40L±anti-LFA-1 or isotype control antibodies during the first week after transplantation. Half of the costimulation blockade-treated recipients had their grafts removed after 8 weeks. The other half was observed up to 5 months. Results. Recipients treated with CTLA4Ig/anti-CD40L/anti-LFA-1 had significantly lower blood glucose and gained more weight than CTLA4Ig/anti-CD40L-treated recipients. CTLA4Ig/anti-CD40L-treated recipients exhibited unstable blood glucose. IPGTT of these recipients revealed a slow recovery to normal blood glucose levels at week 4. In comparison, CTLA4Ig/anti-CD40L/anti-LFA-1 treated recipients exhibited a significantly superior glucose clearance. CTLA4Ig/anti-CD40L±anti-LFA-1 treated recipients did not produce anti-pig IgG, whereas control antibody-treated mice did. CD4+ T cells from costimulation blockade-treated recipients proliferated less than CD4+ T cells from control antibody-treated mice when co-cultured with syngeneic antigen presenting cells loaded with pig islet antigens. Conclusions. CTLA4Ig/anti-CD40L/anti-LFA-1-treated recipients had superior islet function compared with CTLA4Ig/anti-CD40L-treated recipients. However, both costimulation blockade regimens led to islet graft acceptance up to 5 months after a 1-week treatment.

Multipotent Mesenchymal Stromal Cells Synergize With Costimulation Blockade in the Inhibition of Immune Responses and the Induction of Foxp3+ Regulatory T Cells

Multipotent mesenchymal stromal cell (MSC) therapy and costimulation blockade are two immunomodulatory strategies being developed concomitantly for the treatment of immunological diseases. Both of these strategies have the capacity to inhibit immune responses and induce regulatory T cells; however, their ability to synergize remains largely unexplored. In order to study this, MSCs from C57BL/6 (H2b) mice were infused together with fully major histocompatibility complex‐mismatched Balb/c (H2d) allogeneic islets into the portal vein of diabetic C57BL/6 (H2b) mice, which were subsequently treated with costimulation blockade for the first 10 days after transplantation. Mice receiving both recipient‐type MSCs, CTLA4Ig, and anti‐CD40L demonstrated indefinite graft acceptance, just as did most of the recipients receiving MSCs and CTLA4Ig. Recipients of MSCs only rejected their grafts, and fewer than one half of the recipients treated with costimulation blockade alone achieved permanent engraftment. The livers of the recipients treated with MSCs plus costimulation blockade contained large numbers of islets surrounded by Foxp3+ regulatory T cells. These recipients showed reduced antidonor IgG levels and a glucose tolerance similar to that of naïve nondiabetic mice. Intrahepatic lymphocytes and splenocytes from these recipients displayed reduced proliferation and interferon‐γ production when re‐exposed to donor antigen. MSCs in the presence of costimulation blockade prevented dendritic cell maturation, inhibited T cell proliferation, increased Foxp3+ regulatory T cell numbers, and increased indoleamine 2,3‐dioxygenase activity. These results indicate that MSC infusion and costimulation blockade have complementary immune‐modulating effects that can be used for a broad number of applications in transplantation, autoimmunity, and regenerative medicine.

Sublethal caspase activation promotes generation of cardiomyocytes from embryonic stem cells.

Generation of new cardiomyocytes is critical for cardiac repair following myocardial injury, but which kind of stimuli is most important for cardiomyocyte regeneration is still unclear. Here we explore if apoptotic stimuli, manifested through caspase activation, influences cardiac progenitor up-regulation and cardiomyocyte differentiation. Using mouse embryonic stem cells as a cellular model, we show that sublethal activation of caspases increases the yield of cardiomyocytes while concurrently promoting the proliferation and differentiation of c-Kit+/α-actininlow cardiac progenitor cells. A broad-spectrum caspase inhibitor blocked these effects. In addition, the caspase inhibitor reversed the mRNA expression of genes expressed in cardiomyocytes and their precursors. Our study demonstrates that sublethal caspase-activation has an important role in cardiomyocyte differentiation and may have significant implications for promoting cardiac regeneration after myocardial injury involving exogenous or endogenous cell sources.