CD20 expression regulates CD37 levels in B-cell lymphoma - implications for immunotherapies.

Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies.

Role of Ang1-7 in renal haemodynamics and excretion in streptozotocin diabetic rats.

The contribution of angiotensin (1-7) (Ang1-7) to control of extrarenal and renal function may be modified in diabetes. We investigated the effects of Ang1-7 supplementation on blood pressure, renal circulation and intrarenal reactivity (IVR) to vasoactive agents in normoglycaemic (NG) and streptozotocin diabetic rats (DM). In Sprague Dawley DM and NG rats, 3 weeks after streptozotocin (60 mg/kg i.p.) or solvent injection, Ang1-7 was administered (400 ng/min) over the next 2 weeks using subcutaneously implanted osmotic minipumps. For a period of 5 weeks, blood pressure (BP), 24 h water intake and diuresis were determined weekly. In anaesthetised rats, BP, renal total and cortical (CBF), outer (OMBF) and inner medullary (IMBF) perfusion and urine excretion were determined. To check IVR, a short-time infusion of acetylcholine or norepinephrine was randomly given to the renal artery. Unexpectedly, BP did not differ between NG and DM, and this was not modified by Ang-1-7 supplementation. Baseline IMBF was higher in NG vs. DM, and Ang1-7 treatment did not change it in NG but decreased it in DM. In the latter, Ang1-7 increased cortical IVR to vasoconstrictor and vasodilator stimuli. IMBF decrease after high acetylcholine dose seen in untreated NG was reverted to an increase in Ang1-7 treated rats. Irrespective of the glycaemia level, Ang1-7 did not modify BP. However, it impaired medullary circulation in DM, whereas in NG it rendered the medullary vasculature more sensitive to vasodilators. Possibly, the medullary hypoperfusion in DM was mediated by Ang1-7 activation of angiotensin AT-1 receptors which are upregulated by hyperglycaemia.

PRDX-1 Supports the Survival and Antitumor Activity of Primary and CAR-Modified NK Cells under Oxidative Stress.

Oxidative stress, caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses, is one of the characteristic features of cancer. Here, we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo, when compared with blood or normal subcutaneous fluid. Therefore, we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells, and by comparing T, B, and NK cells' sensitivities to redox stress and their antioxidant capacities, we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However, the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore, we genetically modified NK cells to stably overexpress PRDX1, which led to increased survival and NK cell activity in redox stress conditions. Finally, we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide, at concentrations detected in the TME, suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors.

Chymase Dependent Pathway of Angiotensin II Generation and Rapeseed Derived Peptides for Antihypertensive Treatment of Spontaneously Hypertensive Rats.

The contribution of chymase, one of the enzymes responsible for angiotensin II generation in non-ACE pathway, remains unclear in the development of hypertension. The aim of the study was to investigate chymase inhibition as potential antihypertensive therapy in spontaneously hypertensive rats (SHR). To block chymase we employed chymostatin, a commercial inhibitor, and new analogues of rapeseed-derived peptides, VWIS and RIY. These simple and easy to obtain peptides not only block chymase, but also possess weak activity to inhibit ACE. This is a first attempt to evaluate the impact of chronic administration of selected inhibitors on blood pressure of SHR in two phases of hypertension. Male SHR (6 or 16 weeks old) were treated daily for two weeks with chymostatin (CH; 2 mg/kg/day), the peptides VWIS (12.5 mg/kg/day) or RIY (7.5 mg/kg/day); control groups received chymostatin solvent (0.15% DMSO in saline) or peptide solvent (saline). The substances were administered intravenously to conscious animals via a chronically cannulated femoral vein. Systolic blood pressure (SBP) was measured by telemetry. Metabolic parameters were measured weekly, and tissue samples were harvested after two weeks of treatment. None of the administered chymase inhibitors affected the development of hypertension in young rats. Only RIY exhibited beneficial properties when administered in the established phase of hypertension: SBP decreased from 165 ± 10 to 157 ± 7 mmHg while the excretion of nitric oxide metabolites increased significantly. The glomerulosclerosis index was lower after RIY treatment in both age groups (significant only in young rats 0.29 ± 0.05 vs 0.48 ± 0.04 in the control group; p < 0.05). Hence, it seems that peptide RIY exhibits some positive effect on renal morphology. The results obtained suggest that the peptide RIY may be a useful tool in the treatment of hypertension, especially in cases when ACE inhibitors are not effective.

Role of chymase in blood pressure control, plasma and tissue angiotensin II, renal Haemodynamics, and excretion in spontaneously hypertensive rats.

Background: Chymase generates angiotensin II (ANG II) independently of angiotensin-converting enzyme in tissues and it contributes to vascular remodeling and development of hypertension, however the exact mechanism of its action is unclear. Methods: Hence, the effects of chymase inhibition were examined in anesthetized spontaneously hypertensive rats (SHR) in two stages of the disease development, ie. pre-hypertensive (SHR7) and with established hypertension (SHR16). Chymostatin, a commercial chymase inhibitor, was infused intravenously alone or in subsequent groups co-infused with captopril. Results: Mean blood pressure (MBP), total renal blood flow (RBF) and ANG II content (plasma and tissues) were measured. In SHR16 chymase blockade significantly decreased MBP (-6%) and plasma (-38%), kidney (-71%) and heart (-52%) ANG II levels. In SHR7 chymostatin did not influence MBP or RBF, but significantly decreased heart ANG II level. Conclusion: Jointly, functional studies and ANG II determinations support the evidence that in SHR chymase can raise plasma ANG II and contribute to blood pressure elevation. We propose that addition of chymase blockade to ACE inhibition could be a promising approach in the treatment of hypertensive patients resistant to therapy with ACE-inhibitors alone.

Further evidence against the role renal medullary perfusion in short-term control of arterial pressure in normotensive and mildly or overtly hypertensive rats.

Earlier evidence from studies of rat hypertension models undermines the widespread view that the rate of renal medullary blood flow (MBF) is critical in control of arterial pressure (MAP). Here, we examined the role of MBF in rats that were normotensive, with modest short-lasting pressure elevation, or with overt established hypertension. The groups studied were anaesthetised Sprague-Dawley rats: (1) normotensive, (2) with acute i.v. norepinephrine-induced MAP elevation, and (3) with hypertension induced by unilateral nephrectomy followed by administration of deoxycorticosterone-acetate (DOCA) and 1% NaCl drinking fluid for 3 weeks. MBF was measured (laser-Doppler probe) and selectively increased using 4-h renal medullary infusion of bradykinin. MAP, renal excretion parameters and post-experiment medullary tissue osmolality and sodium concentration were determined. In the three experimental groups, baseline MAP was 117, 151 and 171 mmHg, respectively. Intramedullary bradykinin increased MBF by 45%, 65% and 70%, respectively, but this was not associated with a change in MAP. In normotensive rats a significant decrease in medullary tissue sodium was seen. The intramedullary bradykinin specifically increased renal excretion of water, sodium and total solutes in norepinephrine-treated rats but not in the two other groups. As previously shown in models of rat hypertension, in the normotensive rats and those with acute mild pressure elevation (resembling labile borderline human hypertension), 4-h renal medullary hyperperfusion failed to decrease MAP. Nor did it decrease in DOCA-salt model mimicking low-renin human hypertension. Evidently, within the 4-h observation, medullary perfusion was not a critical determinant of MAP in normotensive and hypertensive rats.

Epoxyeicosatrienoic Acid Analog and 20-HETE Antagonist Combination Prevent Hypertension Development in Spontaneously Hypertensive Rats.

Numerous studies indicate a significant role for cytochrome P-450-dependent arachidonic acid metabolites in blood pressure regulation, vascular tone, and control of renal function. Epoxyeicosatrienoic acids (EETs) exhibit a spectrum of beneficial effects, such as vasodilatory activity and anti-inflammatory, anti-fibrotic, and anti-apoptotic properties. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that inhibits sodium reabsorption in the kidney. In the present study, the efficiency of EET-A (a stable analog of 14,15-EET) alone and combined with AAA, a novel receptor antagonist of 20-HETE, was tested in spontaneously hypertensive rats (SHR). Adult SHR (16 weeks old) were treated with two doses of EET-A (10 or 40 mg/kg/day). In the following experiments, we also tested selected substances in the prevention of hypertension development in young SHR (6 weeks old). Young rats were treated with EET-A or the combination of EET-A and AAA (both at 10 mg/kg/day). The substances were administered in drinking water for 4 weeks. Blood pressure was measured by telemetry. Once-a-week observation in metabolic cages was performed; urine, blood, and tissue samples were collected for further analysis. The combined treatment with AAA + EET-A exhibited antihypertensive efficiency in young SHR, which remained normotensive until the end of the observation in comparison to a control group (systolic blood pressure, 134 ± 2 versus 156 ± 5 mmHg, respectively; p < 0.05). Moreover the combined treatment also increased the nitric oxide metabolite excretion. Considering the beneficial impact of the combined treatment with EET-A and AAA in young rats and our previous positive results in adult SHR, we suggest that it is a promising therapeutic strategy not only for the treatment but also for the prevention of hypertension.

Polyprenol-Based Lipofecting Agents for In Vivo Delivery of Therapeutic DNA to Treat Hypertensive Rats.

Development of efficient vectors for transfection is one of the major challenges in genetic engineering. Previous research demonstrated that cationic derivatives of polyisoprenoids (PTAI) may serve as carriers of nucleic acids. In the present study, the effectiveness of two PTAI-based formulations (PTAI-6-8 and 10-14) was investigated and compared to the commercial reagents. The purpose of applied gene therapy was to enhance the expression of vascular endothelial growth factor (VEGF-A) in the renal medulla of spontaneously hypertensive rats (SHR) and to test its potential as a novel antihypertensive intervention. In the first part of the study (in vitro), we confirmed that PTAI-based lipoplexes efficiently transfect XC rat sarcoma cells and are stable in 37 °C for 7 days. In the in vivo experiments, we administered selected lipoplexes directly to the kidneys of conscious SHR (via osmotic pumps). There were no blood pressure changes and VEGF-A level in renal medulla was significantly higher only for PTAI-10-14-based formulation. In conclusion, despite the promising results, we were not able to achieve VEGF-A expression level high enough to verify VEGF-A gene therapy usefulness in SHR. However, results of our study give important indications for the future development of PTAI-based DNA carriers and kidney-targeted gene delivery.

Combined treatment with epoxyeicosatrienoic acid analog and 20-hydroxyeicosatetraenoic acid antagonist provides substantial hypotensive effect in spontaneously hypertensive rats.

OBJECTIVES: The global morbidity and mortality related to hypertension and associated disorders increases continuously and novel therapeutic strategies are still in high demand. Increasing evidence suggests the important role in blood pressure regulation of cytochrome P-450-dependent metabolites of arachidonic acid. Epoxyeicosatrienoic acids (EETs) induce vasodilation and natriuresis, and have renoprotective and anti-inflammatory properties. 20-HETE is an arachidonic acid metabolite with both prohypertensive and antihypertensive activities. To explore the pathophysiological role of arachidonic acid metabolites in more detail, we examined the antihypertensive efficiency of EET-A, a stable analog of 14,15-EET, and of AAA, a novel antagonist of the 20-HETE receptors. METHODS: Male spontaneously hypertensive rats (SHR) were treated for 5 weeks with EET-A, AAA or the combination; age-matched untreated SHR and normotensive Wistar-Kyoto rats served as controls. EET-A and AAA were administered in drinking water at 10 mg/kg/day each. SBP was measured by telemetry and urine, blood, and tissue samples were collected for relevant analyses. RESULTS: EET-A or AAA given alone had no significant effect on SHR blood pressure. In contrast, combined treatment with AAA and EET-A was significantly antihypertensive, causing a decrease in SBP from 180 ±â€Š3 to 160 ±â€Š5 mmHg (P < 0.05). Additionally, the combined treatment attenuated cardiac hypertrophy, decreased kidney ANG II level, increased natriuresis, and increased the excretion of nitric oxide metabolites. CONCLUSION: Considering the beneficial impact of the combined treatment with EET-A and AAA on SHR blood pressure and cardiovascular and renal function, we suggest that the treatment is a promising therapeutic strategy for human hypertension.

Evidence against a crucial role of renal medullary perfusion in blood pressure control of hypertensive rats.

KEY POINTS: The development of new effective methods of treating arterial hypertension is hindered by uncertainty regarding its causes. According to one widespread concept hypertension is caused by abnormal blood circulation in the kidney, specifically by reduction of blood flow through the kidney medulla; however, this causal relationship has never been rigorously verified. We investigated whether in rats with three different forms of experimental hypertension prolonged selective elevation of renal medullary blood flow using local infusion of the vasodilator bradykinin would lower arterial pressure. We found that increasing medullary blood flow by almost 50% did not result in alleviation of hypertension, which argues against a causal role of such changes in the control of arterial pressure and suggests that attempts at improving renal medullary circulation are not likely to be a promising approach to combating hypertension. ABSTRACT: The crucial role of renal medullary blood flow (MBF) in the control of arterial pressure (MAP) has been widely accepted but not rigorously verified. We examined the effects of experimental selective MBF elevation on MAP, medullary tissue hypertonicity and renal excretion in hypertensive rats. We used three hypertensive rat models: (1) rats with hypertension induced by chronic angiotensin II infusions (AngII model), (2) rats with hypertension induced by unilateral nephrectomy followed by high salt diet (HS/UNX), and (3) spontaneously hypertensive rats (SHR). In acute experiments, MBF (laser-Doppler measurement) was selectively increased with an intramedullary infusion of bradykinin (Bk) at 0.27 mg h-1  kg-1 BW over 4 h. MAP, renal artery blood flow (Transonic probe) and renal excretion parameters were measured simultaneously. In chronic studies with AngII and HS/UNX rats, Bk was infused over 2 weeks and MAP (telemetry probe) and renal excretion were repeatedly determined. In acute studies, with AngII, SHR and HS/UNX groups, Bk infusion caused a 47% increase in MBF (P < 0.01-0.001), whereas solvent infusion was without effect. During the experiments MAP decreased slightly and to the same extent with Bk and solvent infusion. Medullary tissue osmolality and [Na+ ] were lower in Bk- than in solvent-infused AngII rats and in SHR. Two weeks of intramedullary Bk infusion tested in AngII and HS/UNX rats did not alter MAP or renal excretion; though in the latter group a significant MBF increase and medullary hypertonicity decrease was observed. Since no decrease in MAP in hypertensive rats was seen with Bk-induced major renal medullary hyperperfusion or with a wash-out of medullary solutes, our data argue against a crucial role of MBF in the pathogenesis of arterial hypertension.

Combined Inhibition of Soluble Epoxide Hydrolase and Renin-Angiotensin System Exhibits Superior Renoprotection to Renin-Angiotensin System Blockade in 5/6 Nephrectomized Ren-2 Transgenic Hypertensive Rats with Established Chronic Kidney Disease.

BACKGROUND/AIMS: We found recently that increasing renal epoxyeicosatrienoic acids (EETs) levels by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, shows renoprotective actions and retards the progression of chronic kidney disease (CKD) in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX). This prompted us to examine if additional protection is provided when sEH inhibitor is added to the standard renin-angiotensin system (RAS) blockade, specifically in rats with established CKD. METHODS: For RAS blockade, an angiotensin-converting enzyme inhibitor along with an angiotensin II type receptor blocker was used. RAS blockade was compared to sEH inhibition added to the RAS blockade. Treatments were initiated 6 weeks after 5/6 NX in TGR and the follow-up period was 60 weeks. RESULTS: Combined RAS and sEH blockade exhibited additional positive impact on the rat survival rate, further reduced albuminuria, further reduced glomerular and tubulointerstitial injury, and attenuated the decline in creatinine clearance when compared to 5/6 NX TGR subjected to RAS blockade alone. These additional beneficial actions were associated with normalization of the intrarenal EETs deficient and a further reduction of urinary angiotensinogen excretion. CONCLUSION: This study provides evidence that addition of pharmacological inhibition of sEH to RAS blockade in 5/6 NX TGR enhances renoprotection and retards progression of CKD, notably, when applied at an advanced stage.

Innovative lipid-based carriers containing cationic derivatives of polyisoprenoid alcohols augment the antihypertensive effectiveness of candesartan in spontaneously hypertensive rats.

Novel lipid-based carriers, composed of cationic derivatives of polyisoprenoid alcohols (amino-prenols, APrens) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), were designed. The carriers, which were previously shown to be nontoxic to living organisms, were now tested if suitable for administration of candesartan, an antihypertensive drug. Spontaneously hypertensive rats (SHR) received injections of candesartan (0.1 mg/kg body weight per day; s.c.) in freshly prepared carriers for two weeks. The rats' arterial pressure was measured by telemetry. Urine and blood collection were performed in metabolic cages. In a separate group of SHR, the pharmacokinetics of the new formulation was evaluated after a single subcutaneous injection. The antihypertensive activity of candesartan administered in DOPE dispersions containing APrens was distinctly greater than that of candesartan dispersions composed of DOPE only or administered in the classic solvent (sodium carbonate). The pharmacokinetic parameters clearly demonstrated that candesartan in APren carriers reached the bloodstream more rapidly and in much greater concentration (almost throughout the whole observation) than the same drug administered in dispersions of DOPE only or in solvent. Serum creatinine (PCr) decreased significantly only in the group receiving candesartan in carriers with APrens (from 0.80 ± 0.04 to 0.66 ± 0.09 mg/dl; p < 0.05), whereas in the other groups PCr remained at the same level after treatment. Moreover, the new derivatives increased the loading capacity of the carriers, which is a valuable feature for any drug delivery system. Taken together, our findings led us to conclude that APrens are potentially valuable components of lipid-based drug carriers.

Effects of chymostatin, a chymase inhibitor, on blood pressure, plasma and tissue angiotensin II, renal haemodynamics and renal excretion in two models of hypertension in the rat.

NEW FINDINGS: What is the central question of this study? We examined, in hypertensive rats, whether the angiotensin-converting enzyme-independent enzymes generating angiotensin II in the tissues modulate blood pressure, peripheral circulation and renal function. What is the main finding and its importance? The results suggest that chymostatin-sensitive enzymes diminish vascular tone in renal and extrarenal vascular beds. Chymase or similar chymostatin-sensitive enzymes have a significant role in the synthesis of angiotensin II in different tissues but do not control blood pressure in the short term, similarly in salt-dependent or Goldblatt-type rat hypertension. In salt-dependent hypertension, chymase blockade protected renal outer medullary perfusion, probably by reducing the angiotensin II content in the kidney. Chymase is presumed to be a crucial enzyme of the non-angiotensin-converting enzyme pathway of angiotensin II (Ang II) generation in tissues, a process involved in vascular remodelling and development of hypertension. We examined the role of chymase in hypertension induced by exposure of uninephrectomized rats to high dietary salt intake (UNX HS) and in the Goldblatt renal artery stenosis (two-kidney, one-clip) model. In acute experiments with anaesthetized rats of either model, chymostatin at 2 mg kg(-1) h(-1) or 0.05% DMSO solvent was infused i.v. Mean arterial blood pressure, heart rate, iliac blood flow (a measure of hindlimb perfusion), total renal blood flow and intrarenal regional perfusion (laser-Doppler technique) were measured continuously, along with the glomerular filtration rate and renal excretion. In both models, chymase blockade distinctly decreased plasma and tissue Ang II without lowering mean blood pressure or consistently altering the other functional parameters measured. Unexpectedly, in Goldblatt hypertensive rats the blockade increased the renal and hindlimb vascular resistances by 51 and 33%, respectively (P < 0.05). In UNX HS hypertensive rats, chymase blockade abolished the solvent-induced decrease in outer medullary blood flow. We conclude that chymase or similar chymostatin-sensitive enzyme(s) has a significant role in the synthesis of Ang II in different tissues but does not participate in short-term control of blood pressure in salt-dependent or Goldblatt-type rat hypertension. In the Goldblatt model, chymase appeared to reduce the renal and hindlimb vascular resistances by an unknown mechanism. In salt-dependent hypertension, chymase blockade protected renal outer medullary perfusion, probably by reducing Ang II content in the kidney.