RESUMEN
Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Amâa common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.
Asunto(s)
Caperuzas de ARN , Vacunas , Animales , Ratones , ARN Mensajero/genética , Caperuzas de ARN/química , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Biosíntesis de Proteínas , MetilaciónRESUMEN
Osteoporosis is a skeletal disease associated with excessive bone turnover. Among the compounds with antiresorptive activity, nitrogen-containing bisphosphonates play the most important role in antiosteoporotic treatment. In previous studies, we obtained two aminomethylidenebisphosphonates-benzene-1,4-bis[aminomethylidene(bisphosphonic)] (WG12399C) acid and naphthalene-1,5-bis[aminomethylidene(bisphosphonic)] (WG12592A) acid-which showed a significant antiproliferative activity toward J774E macrophages, a model of osteoclast precursors. The aim of these studies was to evaluate the antiresorptive activity of these aminobisphosphonates in ovariectomized (OVX) Balb/c mice. The influence of WG12399C and WG12592A administration on bone microstructure and bone strength was studied. Intravenous injections of WG12399C and WG12592A bisphosphonates remarkably prevented OVX-induced bone loss; for example, they sustained bone mineral density at control levels and restored other bone parameters such as trabecular separation. This was accompanied by a remarkable reduction in the number of TRAP-positive cells in bone tissue. However, a significant improvement in the quality of bone structure did not correlate with a parallel increase in bone strength. In ex vivo studies, WG12399C and WG12592A remarkably bisphosphonates reduced osteoclastogenesis and partially inhibited the resorptive activity of mature osteoclasts. Our results show interesting biological activity of two aminobisphosphonates, which may be of interest in the context of antiresorptive therapy.
Asunto(s)
Hueso Esponjoso , Diferenciación Celular/efectos de los fármacos , Difosfonatos/farmacología , Osteoclastos/metabolismo , Osteoporosis Posmenopáusica , Animales , Hueso Esponjoso/metabolismo , Hueso Esponjoso/patología , Línea Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Osteoporosis Posmenopáusica/tratamiento farmacológico , Osteoporosis Posmenopáusica/metabolismo , Osteoporosis Posmenopáusica/patología , OvariectomíaRESUMEN
Vascular endothelial dysfunction and platelet activation play a key role in tumor metastasis, and therefore, both of these processes are considered important therapeutic targets in cancer. The aim of our studies was to analyze antimetastatic activity of combination therapy using nitric oxide donor DETA/NO and antiplatelet drug clopidogrel. Nitric oxide acts as a vasoprotective mediator, while clopidogrel inhibits ADP-mediated platelet aggregation. 4T1-luc2-tdTomato cell line transplanted intravenously (i.v.) and 4T1 cell line transplanted orthotopically were used as metastatic mammary gland cancer models. Moreover, antiaggregation action of compounds was tested ex vivo on the blood samples taken from breast cancer patients. We have shown that in selected dosage regimes, DETA/NO combined with clopidogrel significantly reduced lung metastatic foci formation in an i.v. model, and such inhibition was transiently observed also in an orthotopic model. The antimetastatic effect was correlated with a significant increase of prostacyclin (PGI2) metabolite and reduction of endothelin-1, sE-selectin, sI-CAM, and TGF-ß plasma levels as well as decreased V-CAM expression on the endothelium. Combination therapy decreased fibrinogen binding to the resting platelets at the early stage of tumor progression (day 14). However, at the later stages (days 21 and 28), the markers of platelet activation were detected (increased JON/A antibody bound, P-selectin level, binding of fibrinogen, and vWf). Decreased aggregation as well as a lower release of TGF-ß were detected in platelets incubated ex vivo with compounds tested from metastatic breast cancer patients. Although combination therapy increases E-cadherin, the increase of N-cadherin and α-SMA in tumor tissue was also observed. The results showed that at the early stages of tumor progression, combined therapy with DETA/NO and clopidogrel improves vasoprotective and antiplatelet activity. However, in advanced tumors, some adverse effects toward platelet activation can be observed.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Donantes de Óxido Nítrico/administración & dosificación , Inhibidores de Agregación Plaquetaria/administración & dosificación , Animales , Neoplasias de la Mama/sangre , Línea Celular Tumoral/trasplante , Clopidogrel/administración & dosificación , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Femenino , Humanos , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Activación Plaquetaria/efectos de los fármacos , Plasma Rico en Plaquetas/efectos de los fármacos , Triazenos/administración & dosificaciónRESUMEN
Macrophages are antigen-presenting cells (APCs) that play a crucial role in the innate immune response and may be involved in both clearance and spread of viruses. Stimulation of macrophages via Toll-like receptors (TLRs) results in activation of nuclear factor κB (NF-κB) and synthesis of proinflammatory cytokines. In this work, we show modulation of proinflammatory NF-κB signaling by a member of the family Poxviridae, genus Orthopoxvirus--ectromelia virus (ECTV)--in RAW 264.7 murine macrophages. ECTV interfered with p65 NF-κB nuclear translocation induced by TLR ligands such as lipopolysaccharide (LPS) (TLR4), polyinosinic-polycytidylic acid (poly(I:C)) (TLR3) and diacylated lipopeptide Pam2CSK4 (TLR2/6). We observed that ECTV modulates phosphorylation of Ser32 of inhibitor of κB (IκBα) and Ser536 of p65. Interference of ECTV with TLR signaling pathways implied that proinflammatory cytokine synthesis was inhibited. Our studies provide new insights into the strategies of proinflammatory signaling modulation by orthopoxviruses during their replication cycle in immune cells. Understanding important immune interactions between viral pathogens and APCs might contribute to the identification of drug targets and the development of vaccines.
Asunto(s)
Citocinas/antagonistas & inhibidores , Virus de la Ectromelia/inmunología , Evasión Inmune , Macrófagos/inmunología , Macrófagos/virología , FN-kappa B/metabolismo , Transducción de Señal , Animales , Línea Celular , Lipopolisacáridos/inmunología , Ratones , Poli I-C/inmunología , Transporte de ProteínasRESUMEN
Chitinase-3-like-1 (CHI3L1), also known as YKL-40, is a glycoprotein linked to inflammation, fibrosis, and cancer. This study explored CHI3L1's interactions with various oligosaccharides using microscale thermophoresis (MST) and AlphaScreen (AS). These investigations guided the development of high-throughput screening assays to assess interference of small molecules in binding between CHI3L1 and biotinylated small molecules or heparan sulfate-based probes. Small molecule binders of YKL-40 were identified in our chitotriosidase inhibitors library with MST and confirmed through X-ray crystallography. Based on cocrystal structures of potent hit compounds with CHI3L1, small molecule probes 19 and 20 were designed for an AS assay. Structure-based optimization led to compounds 30 and 31 with nanomolar activities and drug-like properties. Additionally, an orthogonal AS assay using biotinylated heparan sulfate as a probe was developed. The compounds' affinity showed a significant correlation in both assays. These screening tools and compounds offer novel avenues for investigating the role of CHI3L1.
Asunto(s)
Quitinasas , Proteína 1 Similar a Quitinasa-3 , Glicoproteínas , Ensayos Analíticos de Alto Rendimiento , Heparitina SulfatoRESUMEN
Breast cancer is one of the major causes of cancerrelated mortality among women worldwide. It metastasizes to distant organs, particularly to bone tissue. Nitrogencontaining bisphosphonates are mainly used as an adjuvant therapy to inhibit skeletalrelated events; however, there is increasing evidence to suggest that these compounds also exert antitumor effects. In previous studies, the authors synthesized two novel aminomethylidenebisphosphonates (BPs), namely benzene1,4bis[aminomethylidene(bisphosphonic)] acid (WG12399C) and naphthalene1,5bis[aminomethylidene(bisphosphonic)] acid (WG12592A). Both BPs exhibited notable antiresorptive activity in a mouse model of osteoporosis. The present study aimed to assess the in vivo anticancer activity of WG12399C and WG12592A in 4T1 breast adenocarcinoma model. WG12399C exerted an antimetastatic effect by reducing the number of spontaneous lung metastases by ~66% in comparison to the control. In the experimental metastasis model of 4T1luc2tdTomato cells, this compound reduced the incidence of tumor metastases in the lungs by approximately half in comparison to the control. Both WG12399C and WG12595A also significantly reduced the size and/or number of bone metastatic foci. Their proapoptotic and antiproliferative activity may, at least in part, explain the observed effects. Incubation with WG12399C induced an almost 6fold increase in caspase3 activity in 4T1 cells. Moreover, cells treated with WG12399C or WG12595A exhibited a 2fold reduction in invasiveness through Matrigel. Furthermore, both the BPs were able to sensitize the 4T1 cells to cytostatics. In summary, the results of the present study indicate that the examined aminomethylideneBPs may be of particular interest in the context of combined treatment in breast cancer therapy.
Asunto(s)
Neoplasias Óseas , Neoplasias Pulmonares , Animales , Ratones , Femenino , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Difosfonatos/farmacología , Neoplasias Pulmonares/secundario , Línea Celular Tumoral , Ratones Endogámicos BALB CRESUMEN
Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here, we investigated the role of arginase 1 (ARG1) in Vκ*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in Ê-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of Vκ*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However, arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether, these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors.
Asunto(s)
Mieloma Múltiple , Ratones , Animales , Bortezomib/farmacología , Bortezomib/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Arginasa/metabolismo , Cardiotoxicidad , Inhibidores de Proteasoma/farmacologíaRESUMEN
Retinol-binding protein 4 (RBP4) is proposed as an adipokine that links obesity and cancer. We analyzed the role of RBP4 in metastasis of breast cancer in patients and in mice bearing metastatic 4T1 and nonmetastatic 67NR mammary gland cancer. We compared the metastatic and angiogenic potential of these cells transduced with Rbp4 (4T1/RBP4 and 67NR/RBP4 cell lines). Higher plasma levels of RBP4 were observed in breast cancer patients with metastatic tumors than in healthy donors and patients with nonmetastatic cancer. Increased levels of RBP4 were observed in plasma, tumor tissue, liver, and abdominal fat. Moreover, the blood vessel network was highly impaired in mice bearing 4T1 as compared to 67NR tumors. RBP4 transductants showed further impairment of blood flow and increased metastatic potential. Exogenous RBP4 increased lung settlement by 67NR and 4T1 cells. In vitro studies showed increased invasive and clonogenic potential of cancer cells treated with or overexpressing RBP4. This effect is not dependent on STAT3 phosphorylation. RBP4 enhances the metastatic potential of breast cancer tumors through a direct effect on cancer cells and through increased endothelial dysfunction and impairment of blood vessels within the tumor.
RESUMEN
Carbon monoxide and nitric oxide are two of the most important vasoprotective mediators. Their downregulation observed during vascular dysfunction, which is associated with cancer progression, leads to uncontrolled platelet activation. Therefore, the aim of our studies was to improve vasoprotection and to decrease platelet activation during progression of mouse mammary gland cancer by concurrent use of CO and NO donors (CORM-A1 and DETA/NO, respectively). Methods: Mice injected intravenously with 4T1-luc2-tdTomato or orthotopically with 4T1 mouse mammary gland cancer cells were treated with CORM-A1 and DETA/NO. Ex vivo aggregation and activation of platelets were assessed in the blood of healthy donors and breast cancer patients. Moreover, we analyzed the compounds' direct effect on 4T1 mouse and MDA-MB-231 human breast cancer cells proliferation, adhesion and migration in vitro. Results: We have observed antimetastatic effect of combination therapy, which was only transient in orthotopic model. During early stages of tumor progression concurrent use of CORM-A1 and DETA/NO demonstrated vasoprotective ability (decreased endothelin-1, sICAM and sE-selectin plasma level) and downregulated platelets activation (decreased bound of fibrinogen and vWf to platelets) as well as inhibited EMT process. Combined treatment with CO and NO donors diminished adhesion and migration of breast cancer cells in vitro and inhibited aggregation as well as TGF-ß release from breast cancer patients' platelets ex vivo. However, antimetastatic effect was not observed at a later stage of tumor progression which was accompanied by increased platelets activation and endothelial dysfunction related to a decrease of VASP level. Conclusion: The therapy was shown to have antimetastatic action and resulted in normalization of endothelial metabolism, diminution of platelet activation and inhibition of EMT process. The effect was more prominent during early stages of tumor dissemination. Such treatment could be applied to inhibit metastasis during the first stages of this process.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Boranos/farmacología , Carbonatos/farmacología , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/patología , Óxido Nítrico/metabolismo , Compuestos Nitrosos/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Boranos/uso terapéutico , Carbonatos/uso terapéutico , Bovinos , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Progresión de la Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Endotelio/efectos de los fármacos , Endotelio/fisiopatología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Humanos , Hidrazinas/farmacología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Animales/irrigación sanguínea , Ratones Endogámicos BALB C , Proteínas de Microfilamentos/metabolismo , Metástasis de la Neoplasia , Óxido Nítrico/farmacología , Compuestos Nitrosos/uso terapéutico , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Factores de TiempoRESUMEN
Calcitriol and its analogues are considered drugs supporting the anticancer treatment of breast cancer and preventing the osteoporosis that results from the development of cancer or from chemotherapy or hormone therapy. Following the orthotopic implantation of 4T1 mammary carcinoma cells into aged ovariectomized (OVX) mice, we evaluated the effects of calcitriol and its two analogues, PRI-2191 and PRI-2205, on metastatic spread and bone homeostasis. Calcitriol and its analogues temporarily inhibited the formation of metastases in the lungs. Unexpectedly, only mice treated with calcitriol analogues showed a deterioration of bone-related parameters, such as bone column density, marrow column density and the CaPO4 coefficient. These findings correlated with an increased number of active osteoclasts differentiated from bone marrow-derived macrophages in mice treated with the analogues. Interestingly, in the tumours from mice treated with PRI-2191 and PRI-2205, the expression of Tnfsf11 (RANKL) was increased. On the other hand, osteopontin (OPN) levels in plasma and tumour tissue, as well as TRAC5b levels in tumours, were diminished by calcitriol and its analogues. Despite a similar action of both analogues towards bone metabolism, their impact on vitamin D metabolism differed. In particular, PRI-2191 and calcitriol, not PRI-2205 treatment significantly diminished the levels of both 25(OH)D3 and 24,25(OH)2D3. In conclusion, though there is evident antimetastatic activity in old OVX mice, signs of increased bone metabolism and deterioration of bone mineralization during therapy with calcitriol analogues were observed.
RESUMEN
Low vitamin D status is considered as a risk factor for breast cancer and has prognostic significance. Furthermore, vitamin D deficiency increases after adjuvant cancer therapy, which alters bone metabolism increasing the risk of osteoporosis. It is now postulated that vitamin D supplementation in breast cancer treatment delays the recurrence of cancer thereby extending survival. We evaluated the impact of calcitriol and its low-calcemic analogs, PRI2191 and PRI2205, on the tumor growth, angiogenesis, and metastasis of 4T1 mouse mammary gland cancer. Gene expression analysis related to cancer invasion/metastasis, realtime PCR, ELISA, western blotting, and histochemical studies were performed. In vitro studies were conducted to compare the effects of calcitriol and its analogs on 4T1 and 67NR cell proliferation and expression of selected proteins. Calcitriol and its analogs increased lung metastasis without influencing the growth of primary tumor. The levels of plasma 17ß-estradiol and transforming growth factor ß (TGFß) were found to be elevated after treatment. Moreover, the results showed that tumor blood perfusion improved and osteopontin (OPN) levels increased, whereas vascular endothelial growth factor (VEGF) and TGFß levels decreased in tumors from treated mice. All the studied treatments resulted in increased collagen content in the tumor tissue in the early step of tumor progression, and calcitriol caused an increase in collagen content in lung tissue. In addition, in vitro proliferation of 4T1 tumor cells was not found to be affected by calcitriol or its analogs in contrast to non-metastatic 67NR cells. Calcitriol and its analogs enhanced the metastatic potential of 4T1 mouse mammary gland cancer by inducing the secretion of OPN probably via host cells. In addition, OPN tumor overexpression prevailed over the decreasing tumor TGFß level and blood vessel normalization via tumor VEGF deprivation induced by calcitriol and its analogs. Moreover, the increased plasma TGFß and 17ß-estradiol levels contributed to the facilitation of metastatic process.
Asunto(s)
Calcitriol/análogos & derivados , Calcitriol/farmacología , Dihidroxicolecalciferoles/farmacología , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/patología , Animales , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Microambiente Tumoral/efectos de los fármacosRESUMEN
Clopidogrel, a thienopyridine derivative with antiplatelet activity, is widely prescribed for patients with cardiovascular diseases. In addition to antiplatelet activity, antiplatelet agents possess anticancer and antimetastatic properties. Contrary to this, results of some studies have suggested that the use of clopidogrel and other thienopyridines accelerates the progression of breast, colorectal, and prostate cancer. Therefore, in this study, we aimed to evaluate the efficacy of clopidogrel and various anticancer agents as a combined treatment using mouse models of breast, colorectal, and prostate cancer. Metastatic dissemination, selected parameters of platelet morphology and biochemistry, as well as angiogenesis were assessed. In addition, body weight, blood morphology, and biochemistry were evaluated to test toxicity of the studied compounds. According to the results, clopidogrel increased antitumor and/or antimetastatic activity of chemotherapeutics such as 5-fluorouracil, cyclophosphamide, and mitoxantrone, whereas it decreased the anticancer activity of doxorubicin, cisplatin, and tamoxifen. The mechanisms of such divergent activities may be based on the modulation of tumor vasculature via factors, such as transforming growth factor ß1 released from platelets. Moreover, clopidogrel increased the toxicity of docetaxel and protected against mitoxantrone-induced toxicity, which may be due to the modulation of hepatic enzymes and protection of the vasculature, respectively. These results demonstrate that antiplatelet agents can be useful but also dangerous in anticancer treatment and therefore use of thienopyridines in patients undergoing chemotherapy should be carefully evaluated.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Inhibidores de Agregación Plaquetaria/farmacología , Ticlopidina/análogos & derivados , Animales , Línea Celular Tumoral , Clopidogrel , Fluorouracilo/farmacología , Humanos , Masculino , Ratones , Neoplasias de la Próstata/patología , Ticlopidina/uso terapéuticoRESUMEN
The discovery of tumor angiogenesis opened a new path in fighting cancer. The approval of different antiangiogenic agents, most targeting vascular endothelial growth factor (VEGF) signaling, has either increased the effectiveness of standard chemotherapy or even replaced it by offering better patient outcomes. However, an increasing number of preclinical and clinical observations have shown that the process of angiogenesis is far from clearly understood. Apart from targeting the VEGF pathway, novel strategies aim to influence other molecular factors that are involved in tumor angiogenesis. In addition, naturally occurring compounds seem to offer additional agents for influencing angiogenesis. The first concept of antiangiogenic therapy aimed to destroy tumor vessels, while it turned out that, paradoxically, antiangiogenic drugs normalized vasculature and as a result offered an improvement in chemotherapeutic delivery. In order to design an effective treatment schedule, methods for detecting the time window of normalization and biomarkers predicting patient response are needed. The initial idea that antiangiogenic therapy would be resistance-free failed to materialize and currently we still face the obstacle of resistance to antiangiogenic therapy.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Animales , Humanos , Neoplasias/irrigación sanguíneaRESUMEN
Endothelial cells accompany the malignant cancer cell in almost every stage of metastatic process which includes: infiltration of tumor cells into the neighboring tissue, transmigration through endothelium (intravasation), survival in the blood stream, and extravasation followed by colonization of the target organ. The blood vessels within the tumor are heterogeneous, highly permeable, and chaotically branched therefore often described as abnormal or dysfunctional. These abnormalities are common for all components of the vessel wall and result from the activity of such factors as hypoxia and chronic growth factor stimulation. In this review, we focus not only on the distinctions in terms of the characteristic and function of tumor endothelial cells (TEC) as compared to normal endothelial cells (NEC), but also on all of these metastasis steps, which are accompanied by endothelial mediated mechanisms. Moreover, some therapeutic approaches directly or indirectly targeting the endothelium are discussed.