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In this study, we present the design, implementation, and successful use of digital droplet PCR (ddPCR) for the monitoring of chimeric antigen receptor T-cell (CAR-T) expansion in patients with B-cell malignancies treated with different CAR-T products at our clinical center. Initially, we designed a specific and highly sensitive ddPCR assay targeting the junction between the 4-1BB and CD3ζ domains of tisa-cel, normalized with RPP30 , and validated it using blood samples from the first tisa-cel-treated patient in Switzerland. We further compared this assay with a published qPCR (quantitative real-time PCR) design. Both assays showed reliable quantification of CAR-T copies down to 20 copies/µg DNA. The reproducibility and precision were confirmed through extensive testing and inter-laboratory comparisons. With the introduction of other CAR-T products, we also developed a corresponding ddPCR assay targeting axi-cel and brexu-cel, demonstrating high specificity and sensitivity with a limit of detection of 20 copies/µg DNA. These assays are suitable for CAR-T copy number quantification across multiple sample types, including peripheral blood, bone marrow, and lymph node biopsy material, showing robust performance and indicating the presence of CAR-T cells not only in the blood but also in target tissues. Longitudinal monitoring of CAR-T cell kinetics in 141 patients treated with tisa-cel, axi-cel, or brexu-cel revealed significant expansion and long-term persistence. Peak expansion correlated with clinical outcomes and adverse effects, as is now well known. Additionally, we quantified the CAR-T mRNA expression, showing a high correlation with DNA copy numbers and confirming active transgene expression. Our results highlight the quality of ddPCR for CAR-T monitoring, providing a sensitive, precise, and reproducible method suitable for clinical applications. This approach can be adapted for future CAR-T products and will support the monitoring and the management of CAR-T cell therapies.

There is increasing evidence that therapy-related acute lymphoblastic leukemia (trALL) resulting from chemo- and/or radiotherapy represents a distinct entity. However, apart from KMT2A rearrangements, which have been repeatedly reported in this subgroup, the relevance of other aberrations remains controversial due to divergent study results and sparse molecular analyses. Within our ALL patient cohort, 15% ( n  = 19/131) met the criteria for trALL with a high proportion of Ph + and KMT2A rearrangements. On the molecular level, the most frequently observed mutation was KMT2D , followed by CDKN2A , KRAS and DNMT3A . No TP53 mutation was detected. Outcome was particularly poor in Ph + trALL compared to Ph+ de novo ALL, which seemed to be mitigated by allogeneic stem cell transplantation. Our findings further define trALL as a distinct entity but highlight the need for further molecular genome sequencing of somatic and germline variants to advance our understanding of trALL.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells 1 . Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies 2 . However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs 2-5 . Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies., (© 2024. The Author(s).)

Background and Objectives: Myasthenia gravis (MG) can in rare cases be an autoimmune phenomenon associated with hematologic malignancies such as chronic lymphocytic leukemia (CLL). It is unclear whether in patients with MG and CLL, the leukemic B cells are the ones directly driving the autoimmune response against neuromuscular endplates., Methods: We identified patients with acetylcholine receptor antibody-positive (AChR + ) MG and CLL or monoclonal B-cell lymphocytosis (MBL), a precursor to CLL, and described their clinical features, including treatment responses. We generated recombinant monoclonal antibodies (mAbs) corresponding to the B-cell receptors of the CLL phenotype B cells and screened them for autoantigen binding., Results: A computational immune cell screen revealed a subgroup of 5/38 patients with MG and 0/21 healthy controls who displayed a CLL-like B-cell phenotype. In follow-up hematologic flow cytometry, 2 of these 5 patients were diagnosed with an MBL. An additional patient with AChR + MG as a complication of manifest CLL presented at our neuromuscular clinic and was successfully treated with the anti-CD20 therapy obinutuzumab plus chlorambucil. We investigated the specificities of expanding CLL-like B-cell clones to assess a direct causal link between the 2 diseases. However, we observed no reactivity of the clones against the AChR, antigens at the neuromuscular junction, or other common autoantigens., Discussion: Our study suggests that AChR autoantibodies are produced by nonmalignant, polyclonal B cells The new anti-CD20 treatment obinutuzumab might be considered in effectively treating AChR + MG., Classification of Evidence: This is a single case study and provides Class IV evidence that obinutuzumab is safe to use in patients with MG., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

In classical hairy cell leukemia (HCL), standard treatments including purine analogs achieve a durable response (up to 90%), but lead to severe immunosuppression and long-lasting depletion of CD4 + T lymphocytes. The BRAF inhibitor vemurafenib is effective in HCL, but its use in first-line treatment is restricted to select clinical situations (e.g. active infection). Its impact on immune function or response to vaccines in HCL is unclear. We treated four HCL patients with vemurafenib during the COVID-19 pandemic and monitored immune reconstitution and response to SARS-CoV-2 immunization. All patients responded to HCL treatment with normalization of peripheral blood counts. No severe infections occurred. As an indication of limited immunosuppression by vemurafenib, stable CD4 + and CD8 + T lymphocyte counts and immunoglobulin levels were observed. Three out of four patients received SARS-CoV-2 vaccination (Pfizer-BioNTech) during treatment with vemurafenib. IgG antibody levels against the spike-protein of SARS-CoV-2 were detected (40-818 AE/ml). Our data suggest that vemurafenib has limited effects on cellular and humoral immune function in HCL, which allows for successful SARS-CoV-2 vaccination. These data support the use of BRAF inhibitors during the current pandemic where continued immune response is necessary for minimizing the COVID-19-related risk of non-vaccinated patients., (© 2022. The Author(s).)

Background: Azathioprine is a widely prescribed drug for patients with chronic inflammatory diseases such as myasthenia gravis or organ transplant recipients. Azathioprine exerts immunosuppressive effects by inhibiting intracellular purine synthesis and reducing the numbers of circulating B and T lymphocytes. Case reports indicate increased risk for serious infections that can occur despite regular measurements of lymphocyte counts during azathioprine therapy., Objective: We sought to comprehensively investigate therapy-associated patient risks and the underlying immune dysfunction of azathioprine use., Methods: Peripheral blood leukocytes were analyzed using single-cell mass and spectral flow cytometry to detect specific effects of azathioprine use on the systemic immune signature. Therapy-associated clinical features were analyzed in 2 independent cohorts of myasthenia gravis patients., Results: Azathioprine therapy selectively induced pronounced CD56 dim CD16 + natural killer cell depletion and concomitant IFN-γ deficiency. Cytokine profiling revealed a specific contraction of classical T H 1 cells during azathioprine treatment. We further observed an increased occurrence of reactivation of endogenous latent herpesviruses in the azathioprine-treated group versus in patients with myasthenia gravis who were not receiving immunomodulatory treatment; this increased occurrence was validated in an independent cohort., Conclusion: Our study highlights the risk of development of adverse events during azathioprine therapy and suggests that natural killer cell monitoring could be valuable in clinical practice., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Next-generation sequencing has greatly advanced the molecular diagnostics of malignant hematological diseases and provides useful information for clinical decision making. Studies have shown that certain mutations are associated with prognosis and have a direct impact on treatment of affected patients. Therefore, reliable detection of pathogenic variants is critically important. Here, we compared four sequencing panels with different characteristics, from number of genes covered to technical aspects of library preparation and data analysis workflows, to find the panel with the best clinical utility for myeloid neoplasms with a special focus on acute myeloid leukemia. Using the Acrometrix Oncology Hotspot Control DNA and DNA from acute myeloid leukemia patients, panel performance was evaluated in terms of coverage, precision, recall, and reproducibility and different bioinformatics tools that can be used for the evaluation of any next-generation sequencing panel were tested. Taken together, our results support the reliability of the Acrometrix Oncology Hotspot Control to validate and compare sequencing panels for hematological diseases and show which panel-software combination (platform) has the best performance., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aim of the Study: Chimeric antigen receptor T (CAR-T) cells are a powerful form of immune-cell therapy for patients with relapsed/refractory B-cell lymphoma and acute B lymphoblastic leukaemia. CAR-T cells have been commercially available in Switzerland since 2018. Because of the complexity and costs of this treatment it is critical to review patient outcomes in real-world settings, to examine whether the promising results from pivotal trials can be reproduced and to identify clinical parameters that determine their efficacy., Methods: Here we present results of a retrospective study analysing outcomes of patients treated with CAR-T cells in a single academic centre in Switzerland during the first two years after commercial approval (BASEC-No. 2020-02271). Cytokine release syndrome (CRS), immune-cell associated neurotoxicity syndrome (ICANS), responses to treatment, ancillary laboratory studies and administrative specifics of CAR-T treatment were examined and are discussed., Results: From October 2018 to August 2020 CAR-T cell therapy was evaluated in 34 patients, mostly with relapsed/refractory aggressive B-cell lymphoma (87% had refractory disease). Thirty-one patients underwent leukapheresis. Three of 31 patients (9.6%) died of rapid disease progression before the CAR-T cell product was delivered, two patients were enrolled into a clinical trial, three patients were not given CAR-T cells for other reasons. Ultimately, 23 patients were infused with a commercial CAR-T cell product and included in this analysis. Fourteen (61%) patients received bridging therapy while waiting for a median of 41 days (range 31-62) for delivery of the CAR-T cell product. Toxicity and severe side effects were rare (CRS >3 in 13%, ICANS > grade 3 in 10% of patients), manageable and resolved completely thereafter. The best overall response rate was 65%, with complete responses in 38% of lymphoma patients. At 12 months postinfusion, 61% of patients were alive and 35% progression free. With a median follow-up of 14 months, 13/23 (56%) patients were alive at the time of writing., Conclusion: CAR-T cell therapy proved to be safe and manageable under adequate hospital conditions. Outcomes resemble results from pivotal trials. The majority of patients was heavily pretreated and refractory at the time of CAR-T cell infusion. Patient selection, time point of leukapheresis, bridging strategies and timing of CAR-T cell infusion may be critical to further improve outcomes.

Elderly patients (EP) of 60 years and above with acute lymphoblastic leukemia (ALL) have a dismal prognosis, but pediatric-inspired chemotherapy and allogeneic stem cell transplantation (allo HCT) are used reluctantly due to limited data and historical reports of high treatment-related mortality in EP. We analyzed 130 adult ALL patients treated at our center between 2009 and 2019, of which 26 were EP (range 60-76 years). Induction with pediatric-inspired protocols was feasible in 65.2% of EP and resulted in complete remission in 86.7% compared to 88.0% in younger patients (YP) of less than 60 years. Early death occurred in 6.7% of EP. Three-year overall survival (OS) for Ph - B-ALL was significantly worse for EP (n = 16) than YP (n = 64) with 30.0% vs 78.1% (p ≤ 0.001). Forty-nine patients received allo HCT including 8 EP, for which improved 3-year OS of 87.5% was observed, whereas EP without allo HCT died after a median of 9.5 months. In Ph + B-ALL, 3-year OS did not differ between EP (60.0%, n = 7) and YP (70.8%, n = 19). Non-relapse mortality and infection rate were low in EP (14.3% and 12.5%, respectively). Our data indicate that selected EP can be treated effectively and safely with pediatric regimens and might benefit from intensified therapy including allo HCT., (© 2022. The Author(s).)

Myasthenia gravis (MG) is an autoimmune disease characterized by impaired neuromuscular signaling due to autoantibodies targeting the acetylcholine receptor. Although its auto-antigens and effector mechanisms are well defined, the cellular and molecular drivers underpinning MG remain elusive. Here, we employed high-dimensional single-cell mass and spectral cytometry of blood and thymus samples from MG patients in combination with supervised and unsupervised machine-learning tools to gain insight into the immune dysregulation underlying MG. By creating a comprehensive immune map, we identified two dysregulated subsets of inflammatory circulating memory T helper (Th) cells. These signature Th CD103 and Th GM cells populated the diseased thymus, were reduced in the blood of MG patients, and were inversely correlated with disease severity. Both signature Th subsets rebounded in the blood of MG patients after surgical thymus removal, indicative of their role as cellular markers of disease activity. Together, this in-depth analysis of the immune landscape of MG provides valuable insight into disease pathogenesis, suggests novel biomarkers and identifies new potential therapeutic targets for treatment.

Background: Cancer immunotherapy via immune-checkpoint inhibition (ICI) by antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and cell death protein 1 (PD-1) have significantly improved the outcome of metastasized melanoma and of a rapidly increasing number of other cancer types. The anti-tumor effect is often accompanied by immune-related adverse events (irAE). Hematological irAE, specifically neutropenia, are rarely observed. However, neutropenia is associated with high morbidity and mortality due to infection complications. Thus, early detection and treatment is crucial., Methods: We present the clinical course of two patients with severe neutropenia after ICI therapy and demonstrate the difficulty of the diagnosis when a comedication of metamizole, a well-known analgesic drug used to treat cancer pain, is present. Further, we provide a comprehensive descriptive and statistical analysis of published data on diagnostics, treatment and infection complication in patients with at least grade 4 neutropenia by a systematic database search., Results: Finally, 34 patients were analyzed, including the two case reports from our cohort. The median onset of neutropenia was 10.5 weeks after first ICI administration (interquartile range: 6 weeks). In 76% (N = 26), a normalization of the neutrophil count was achieved after a median duration of neutropenia of 13 days. In a subsample of 22 patients with detailed data, the infection rate was 13%, proven by positive blood culture in 3 cases, but 68% (N = 15) presented with fever > 38 °C. Treatment regime differed relevantly, but mainly included G-CSF and intravenous corticosteroids. Death was reported in 14 patients (41%), 3 of whom (9%) were associated with hematological irAE but only two directly associated with neutropenia., Conclusion: With an increasing number of cancer patients eligible to ICI therapy, the incidence of severe hematological toxicities may rise substantially over the next years. Clinicians working in the field of cancer immune therapies should be aware of neutropenia as irAE to provide immediate treatment.

The clinical and biological relevance of a paraprotein that newly arises after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in non-myeloma patients is unknown. In this study, the incidence, the course, and the clinical impact of paraproteins found after allo-HSCT were investigated in a cohort of 383 non-myeloma patients. Paraproteinemia after allo-HSCT was more frequent (52/383 patients, 14%) than the reported incidence of monoclonal gammopathy of unknown significance (MGUS) in age-matched healthy subjects and, in contrast to MGUS, did not correlate with age. In most patients (32/52, 62%), the paraprotein appeared transiently within the first year after allo-HSCT with a median duration of 6.0 months. Post-allo-HSCT paraproteinemia was significantly associated with graft versus host disease (GvHD) and correlated with a survival benefit within the first year, but not after five years following allo-HSCT. Importantly, patients with post-allo-HSCT paraproteinemia did not progress into a plasma cell myeloma as observed for MGUS inferring a distinct pathogenic mechanism. Skewing of lymphocyte subpopulations and alterations in cytokine levels in GvHD may explain the expansion of a specific plasma cell subset in non-myeloma patients undergoing allo-HSCT. Our data suggests that paraproteinemia after allo-HSCT is a reactive phenomenon rather than the consequence of clonal plasma cell transformation., Competing Interests: CONFLICTS OF INTEREST None.

Although the molecular pathways that cause acute myeloid leukemia (AML) are increasingly well understood, the pathogenesis of peripheral blood cytopenia, a major cause of AML mortality, remains obscure. A prevailing assumption states that AML spatially displaces nonleukemic hematopoiesis from the bone marrow. However, examining an initial cohort of 223 AML patients, we found no correlation between bone marrow blast content and cytopenia, questioning the displacement theory. Measuring serum concentration of thrombopoietin (TPO), a key regulator of hematopoietic stem cells and megakaryocytes, revealed loss of physiologic negative correlation with platelet count in AML cases with blasts expressing MPL, the thrombopoietin (scavenging) receptor. Mechanistic studies demonstrated that MPL hi blasts could indeed clear TPO, likely therefore leading to insufficient cytokine levels for nonleukemic hematopoiesis. Microarray analysis in an independent multicenter study cohort of 437 AML cases validated MPL expression as a central predictor of thrombocytopenia and neutropenia in AML. Moreover, t(8;21) AML cases demonstrated the highest average MPL expression and lowest average platelet and absolute neutrophil counts among subgroups. Our work thus explains the pathophysiology of peripheral blood cytopenia in a relevant number of AML cases., (© 2016 by The American Society of Hematology.)

Hemoglobin is considered a potentially toxic molecule when released from erythrocytes during hemolysis, inflammation, or tissue injury. The mechanisms of toxicity involve reduced nitric oxide bioavailability and oxidative processes both occurring at the heme prosthetic groups. When the endogenous oxidant H(2)O(2) reacts with Hb, transient radicals are generated during the peroxidative consumption of H(2)O(2). If not neutralized, these radicals can lead to tissue toxicity. The net biologic effect of extracellular Hb in an H(2)O(2)-rich environment will therefore be determined by the balance of H(2)O(2) decomposition (potential protective effect) and radical generation (potential damaging effect). Here we show that Hb can protect different cell types from H(2)O(2)-mediated cell death and the associated depletion of intracellular glutathione and ATP. Importantly, Hb blunts the transcriptional oxidative-stress response induced by H(2)O(2) in human vascular smooth muscle cells (VSMCs). Based on spectrophotometric and quantitative mass spectrometry analysis, we suggested a novel mechanism in which Hb redox-cycles H(2)O(2) and simultaneously internalizes the radical burden, with irreversible structural globin changes starting with specific amino acid oxidation involving the heme proximate betaCys93 and ultimately ending with protein precipitation. Our results suggest that complex interactions determine whether extracellular Hb, under certain circumstances, acts a protective or a damaging factor during peroxidative stress conditions.

Objective: To determine effects of dietary fat content on vascular responses in different conduit arteries in mice., Methods and Procedures: Vascular responses to reactive oxygen species (ROS)/hydroxyl radical (.OH), acetylcholine (ACh), endothelin-1 (ET-1), and angiotensin II (Ang II) were determined in carotid and femoral arteries of C57BL/6J mice fed with diets varying in fat content (low fat (LF), 12.3%; high fat (HF), 41%; and very high fat (VHF), 58% (kcal from fat)) for 15 weeks, beginning at 4 weeks of age., Results: In precontracted rings of carotid and femoral artery, ROS/.OH-induced a rapid, transient vasodilation. In the carotid, but not in femoral artery, ROS/.OH-induced dilation increased with increasing dietary fat intake (P < 0.05 vs. LF diet), while contractile responses to ROS/.OH remained unaffected. In femoral arteries, ROS/.OH-induced contractions were reversed into relaxations after both HF and VHF diet (P < 0.05 vs. LF diet). Both ET-1 and Ang II induced strong contractions in the femoral artery that were unaffected by dietary fat intake. In contrast, in the carotid artery Ang II-induced contraction was attenuated after HF and VHF diets (P < 0.005 vs. LF diet), whereas ET-1-induced vasoconstriction was significantly increased (P < 0.05 VHF vs. LF and HF). Treatment with VHF diet enhanced ACh-mediated endothelium-dependent relaxation only in the femoral artery (P < 0.05 vs. HF)., Discussion: These findings demonstrate that dietary fat content has regional and distinct effects on vascular function in different vascular beds. The data also suggest the possibility that in selected conduit arteries ROS-dependent vasodilator mechanisms become activated in response to increased dietary fat intake.

Reactive oxygen species (ROS) and endothelin-1 (ET-1) contribute to vascular pathophysiology in obesity. In this context, whether ET-1 modulates hydroxyl radical (*OH) formation and the function of ROS/*OH in obesity is not known. In the present study, formation and function of ROS, including *OH, were investigated in the aorta of lean and leptin-deficient obese ob/ob mice. Hydroxyl radical formation was detected ex vivo using terephthalic acid in intact aortic rings and the involvement of ROS in ET-1-mediated vasoreactivity was analyzed using the antioxidant EPC-K1, a combination of alpha-tocopherol and ascorbic acid. Generation of either *OH, *O(2)(-), and H(2)O(2) was strongly inhibited by EPC-K1 (all P < 0.05). In obese mice, basal vascular *OH formation and ROS activity were reduced by 3-fold and 5-fold, respectively (P < 0.05 vs. lean). ET-1 markedly enhanced *OH formation in lean (6-fold, P < 0.05 vs. untreated) but not in obese mice. Obesity increased ET-1-induced contractions (P < 0.05 vs. lean), and ROS scavenging further enhanced the response (P < 0.05 vs. untreated). Exogenous ROS, including *OH caused stronger vasodilation in obese animals (P < 0.05 vs. lean), whereas endothelium-dependent relaxation was similar between lean and obese animals. In conclusion, we present a sensitive method allowing ex vivo measurement of vascular *OH generation and provide evidence that ET-1 regulates vascular *OH formation. The data indicate that in obesity, vascular formation of ROS, including *OH is lower, whereas the sensitivity to ROS is increased, suggesting a novel and important role of ROS, including *OH in the regulation of vascular tone in disease status associated with increased body weight.

Objective: Angiotensin II (Ang II), endothelin-1 (ET-1) and reactive oxygen species (ROS) have been implicated in the development of pathologic changes associated with obesity including hypertension and atherosclerosis. The aim of this study was to investigate the effects of dietary fat content on vasoreactivity and receptor expression at the level of gene and protein expression., Methods: C57BL/6 mice were fed diets of normal (Control, 12.3% kcal from fat), high (HF, 41% kcal from fat) and very high (VHF, 58% kcal from fat) fat content for 15 weeks. Glucose tolerance tests were performed, and aortic rings were exposed to ET-1 (0.01-300 nM) and Ang II (100 nM) in the presence of L-nitro-arginine-methyl ester (L-NAME; 300 microM). Gene and protein expressions of angiotensin and endothelin receptors were examined by real-time PCR and immunoblotting, respectively. The effects of diet on responses to acetylcholine (ACh 0.1-300 microM), in the absence or presence of L-NAME, and to exogenous ROS/.OH were also investigated., Results: Both high fat diets similarly impaired glucose tolerance (P<0.05). Increasing dietary fat augmented contractions to Ang II in a step-wise manner (P<0.05). Conversely, increasing dietary fat had no effect on contractions to ET-1. Exposure to ROS/.OH resulted in a rapid vasodilation that was markedly augmented in a step-wise manner with increasing dietary fat (P<0.05). Endothelium-dependent relaxation to ACh was unaffected whereas vasoconstriction to high concentrations of ACh was enhanced in VHF animals (P<0.05 vs. control). Gene expression of the AT(1B) receptor was increased in the aorta of VHF mice, and aortic ET(A) receptor protein expression was increased after both high fat diets., Conclusions: These findings demonstrate that changes in dietary fat intake modulate vascular reactivity in response to Ang II and ROS, as well as expression of vascular angiotensin and endothelin receptors. Dietary fat intake may thereby directly affect cardiovascular risk.

We previously reported that angiotensin II (AngII)-induced vasoconstriction involves activation of cyclooxygenase (COX) in murine aorta and carotid artery. The aim of this study was to investigate the roles of early aging and COX in AngII-induced vasoconstriction in different vascular beds. Aortic, carotid, renal, and femoral artery rings of 19- and 34-week-old C57BL/6 mice were pretreated with the nitric oxide synthase inhibitor L-NAME (300 micromol/L) to exclude effects of NO. Contractions to AngII (100 nmol/L) were recorded in the presence or absence of meclofenamate (10 micromol/L), a nonselective COX inhibitor. The results indicate a pronounced heterogeneity in the vascular responsiveness to AngII. Renal and femoral artery rings showed stronger contractions than aorta or carotid artery (P < 0.01 for both). In all vessels of young animals COX inhibition with meclofenamate only partially blocked vasoconstriction to AngII, whereas contractions were completely abolished in the aorta and carotid artery of older mice (P < 0.05 vs untreated for both). These data demonstrate that COX determines AngII-induced vasoconstriction in the mouse aorta and carotid artery during the early physiological aging process, independent of endothelial NO bioactivity. AngII-induced vasoconstriction in vessels more distal to the heart such as femoral and renal arteries is only in part mediated by COX-dependent mechanisms that remain unaffected by early aging.

Although endothelin (ET)-1 is one of the strongest known vasoconstrictors in most species, we and others have previously found that it is only weakly effective in the mouse aorta. The aim of this study was to further investigate vasoactive effects of ET-1 in vascular beds generally known to be particularly sensitive to ET-1, such as the renal artery. Experiments were performed to determine the vasoconstrictor responses in the thoracic aorta, and in the carotid, femoral, and renal arteries. Isolated vascular rings of C57BL/6 adult male mice (35-40 weeks of age) were exposed to ET-1 (0.01-300 nM) in the presence of the nitric oxide synthase inhibitor l-NAME (0.3 mM) to exclude effects of nitric oxide. Vessels from different vascular beds demonstrated distinct patterns in potency of the contractions to ET-1 and the dynamics of the responses. The maximal contraction to ET-1 was strong and significantly greater in the femoral (105 +/- 7% KCl) and renal artery (62 +/- 7% KCl) than in the carotid artery or the aorta (P < 0.05). The dynamics of the contractile response to ET-1 varied between the different vessels: the renal artery showed a rapid vasoconstriction, followed by a near complete loss of tension, whereas in the aorta, carotid, and femoral artery, vasoconstriction was more sustained. In conclusion, the data demonstrate that mouse femoral and renal arteries exhibit strong contractions in response to ET-1 compared with aorta and carotid artery, and that contractile dynamics differ markedly between arterial vascular beds. These findings may be important for studying the effects of endothelin in mouse models of human disease.