Fundus lesions in malignant hypertension. III. Arterial blood pressure, biochemical, and fundus changes

Malignant (accelerated) renovascular arterial hypertension was produced in 57 adult rhesus monkeys by clamping the renal artery (one-kidney model in 25 animals and two-kidney model in 32). The animals were investigated before renal artery clamping and serially thereafter by recording systolic arterial blood pressure (BP), biochemical changes, and changes in the fundus of the eye; the latter was evaluated by ophthalmoscopy, stereoscopic color fundus photography, and fluorescein fundus angiography. All of the animals developed arterial hypertension. The data on BP, biochemical, and fundus findings were analyzed and correlated. The findings of this study clearly showed that the various fundus lesions seen in these hypertensive animals fall into three distinct categories: (1) hypertensive retinopathy, (2) hypertensive choroidopathy, and (3) hypertensive optic neuropathy. The appearance of the retinopathy was significantly earlier than that of the choroidopathy or optic neuropathy (P less than 0.01), but the difference between the times of appearance of the choroidopathy and neuropathy was not significant. There was no significance in the order in which the three types of fundus changes reached their maximum severity. There was no significant difference between the mean BPs when the retinopathy, choroidopathy, or optic neuropathy first appeared, nor between the BPs at the time of their appearance and at the time when they were most marked. In monkeys of the one-kidney model, the rise in BP developed significantly (P = 0.01) faster and the fundus lesions appeared significantly (P = 0.00001) earlier than in those with the two-kidney model.     

Mechanism of voltage sensing in Ca2+- and voltage-activated K+ (BK) channels

In neurosecretion, allosteric communication between voltage sensors and Ca²⁺ binding in BK channels is crucially involved in damping excitatory stimuli. Nevertheless, the voltage-sensing mechanism of BK channels is still under debate. Here, based on gating current measurements, we demonstrate that two arginines in the transmembrane segment S4 (R210 and R213) function as the BK gating charges. Significantly, the energy landscape of the gating particles is electrostatically tuned by a network of salt bridges contained in the voltage sensor domain (VSD). Molecular dynamics simulations and proton transport experiments in the hyperpolarization-activated R210H mutant suggest that the electric field drops off within a narrow septum whose boundaries are defined by the gating charges. Unlike Kv channels, the charge movement in BK appears to be limited to a small displacement of the guanidinium moieties of R210 and R213, without significant movement of the S4.

Excitable tissues accomplish their signaling functions thanks in part to the interplay of several voltage-sensitive ion channels (1–6). Hence, to understand these processes, it is crucial to establish how voltage-sensitive ion channels sense changes in the electric field across the membrane, an issue that has been a matter of extensive study and intense debate for decades. The most widely accepted mechanism proposes the existence of voltage-sensor domains (VSDs), modules that undergo two or more discrete conformational states in response to changes in the membrane voltage. The simplest model considers two states: active (A), which promotes pore opening, and resting (R), which promotes channel closing. To accomplish its function, VSDs contain voltage-sensitive particles, which move in response to changes in the electric field. This movement triggers the interconversion between the two discrete conformational states. These voltage-sensing particles are typically the guanidine groups of arginine residues within the S4 transmembrane segment, which undergo a combination of rotational, translational, and tilting movement in response to changes in membrane voltage (7–14).The large-conductance Ca²⁺- and voltage-activated K⁺ (BK) channels have a wide distribution in mammalian tissues (15–18), where they participate in a diversity of physiological processes. Their malfunction is often related to diverse pathological conditions (19, 20). BK channel open probability is independently regulated by membrane depolarization and intracellular Ca²⁺ concentration (21, 22), each stimulus being detected by specialized modules. Like other voltage-sensitive K⁺ (Kv) channels, BK is an homotetramer in which each of its α subunits consists of a pore domain (PD; S5-S6 transmembrane segments), a voltage-sensing domain (VSD; S1–S4 transmembrane segments) containing a positively charged S4, and a cytosolic C-terminal regulatory domain, which contains the Ca²⁺-binding sites (23, 24). Also, like some members of other K⁺ channel families (25, 26), the VSD and PD of BK are non–domain swapped (23, 24). BK channels display some distinctive structural and functional features: Despite sharing the selectivity filter sequence with Kv channels, BK unitary conductance and selectivity are exquisitely high (27–30). The BK α subunit has an additional transmembrane segment S0 [therefore, its N terminus faces the extracellular medium (31)], and the voltage sensitivity in BK channels is significantly lower than that of Kv channels, presumably because of their lower number of gating charges (32).Although thoroughly studied, research into BK VSD and its voltage dependence has faced several technical obstacles. The relatively small gating charge per channel (32) and the large conductance of the BK pore makes isolating of the gating currents from the ionic currents a tough experimental challenge. In addition, because mutations of VSD residues can produce very large shifts in both the gating charge-voltage (Q(V)) and the conductance-voltage G(V)) relationships (33), it is necessary to use extreme voltages to accurately measure the voltage dependence of some mutants. Consequently, the identification of BK gating charges has been addressed by using indirect approaches (33, 34). The combination of electrophysiology measurements and kinetic modeling suggests a decentralized VSD in the BK channel, where four charged residues (D153 and R167 in S2, D186 in S3, and R213 in S4) act as voltage sensor particles (33). A recent report of the atomistic cryo-electron microscopy (cryo-EM) structures of the human BK channel and its homolog in Aplysia californica (AcSlo) revealed minor structural differences between the VSD in both the Ca²⁺-bound (open pore) and the Ca²⁺-unbound (closed pore) conformations (23, 24, 35). This result can be explained if the conformational changes of the BK VSD upon activation are small compared to those that occur during the activation of other channels, such as HCN channels (12–14).In this study, we identified voltage-sensing particles in the BK channel by using a direct functional approach, involving gating of current measurements and analysis of the Q(V) curves spanning 800 mV in the voltage axis. Systematic neutralization of the individual charged residues in the VSD (S1–S4) revealed that only the neutralization of two arginines in S4 (R210 and R213) changed the voltage dependence of the Q(V) curves. Neutralization of other VSD charges point to roles in tuning of the half-activation voltage of the VSD and its allosteric coupling with the PD. Molecular dynamics (MD) simulations based on the cryo-EM structures of the human BK channel (35) as templates suggested that R210 and R213 lie in a very narrow septum separating intra- and extracellular water-filled vestibules. This interpretation is consistent with the robust hyperpolarization-activated proton currents generated when R210 is mutated to the protonable amino acid histidine. Overall, our results point to a unique and distinctive mode of activation in BK: In contrast to Kv channels, where positive charges move one by one through a charge transfer center (absent in BK channels) that spans the entire electric field (36, 37), charge movement in BK channels is limited to the small displacement of R210 and R213, which itself constitutes a narrow septum where the electric field drops.     

Evolution of thrombolytic therapy in patients with HeartWare ventricular assist device thrombosis: a single-institutional experience

Open in a separate window OBJECTIVESPump thrombosis remains a major challenge in heart failure patients with left ventricular HeartWare assist device. Current International Society for Heart and Lung Transplantation recommendations favour surgical pump exchange over lysis because safety and efficacy of lysis has been controversially reported. This study summarizes our experience on our HeartWare thrombosis prevention strategy as well as thrombolysis through implementation of our institutional standardized HeartWare assist device protocol.METHODSOutcomes of all HeartWare thrombosis patients admitted between 2010 and 2020 were analysed. Thrombolysis therapy using tissue plasminogen activator was used as the first-line therapy in this study and thrombolysis therapy efficacy was defined as freedom from stroke, bleeding, recurrent HeartWare assist device thrombosis or surgical device exchange within 30 days after lysis application.RESULTSA total of 507 patients have been included in this study and 66 patients (13%) collectively developed a first HeartWare-thrombosis after a median of 12 months (8–22 months) after HeartWare implantation. Forty patients were treated with unstandardized lysis, of whom 7 patients had thrombolysis associated complications, such as incomplete thrombus resolution requiring surgical pump exchange in 4 patients, but also intracranial haemorrhage occurring in 3 patients. Three patients died in the non-protocol group. Eight device thrombosis patients were treated according to our protocol, showing no lysis-associated complication.CONCLUSIONSDespite current recommendations, preferring surgical HeartWare pump exchange in thrombosis, thrombolysis therapy for first HeartWare thrombosis can be safe and effective in a standardized protocol setting, including anticoagulation adjustment and intensified blood pressure control management.     

Extracellular truncations of h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5, lead to ligand-independent activation

The hypothesis that extracellular truncation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony- stimulating factor, and IL-5 (h beta c) can lead to ligand-independent activation was tested by infecting factor-dependent hematopoietic cell lines with retroviruses encoding truncated forms of h beta c. A truncation, resembling that in v-Mpl, and retaining 45 h beta c-derived extracellular residues, led to constitutive activation in the murine myeloid cell line, FDC-P1. However, infection of cells with retrovirus encoding a more severely truncated receptor, retaining only 7 h beta c- derived extracellular residues, did not confer factor independence on these cells. These experiments show that truncation activates the receptor and define a 37-amino acid segment of h beta c (H395-A431) which contains two motifs conserved throughout the cytokine receptor superfamily (consensus Y/H XX R/Q VR and WSXWS), as essential for factor-independent signaling. The mechanism of activation was also investigated in less severe truncations. A receptor that retains the entire membrane-proximal domain (domain 4) also conferred factor independent growth on FDC-P1 cells; however, a retrovirus encoding a truncated form of h beta c having two intact membrane proximal domains did not have this ability, suggesting that domain 3 may have an inhibitory role in h beta c. The ability of these receptors to confer factor independence was cell specific as demonstrated by their inability to confer factor-independent growth when introduced into the murine IL-3-dependent pro-B cell line BaF-B03. These results are consistent with a model in which activation requires unmasking of an interactive receptor surface in domain 4 and association with a myeloid- specific receptor or accessory component. We suggest that in the absence of ligand intramolecular interactions prevent inappropriate signaling.     

Diabetes and cancer: Looking at the multiligand/RAGE axis

The association between diabetes and hyperglycemia and the associated increased risk of several solid and hematologic malignancies has been the subject of investigation for many years.Although the association is not fully understood,current knowledge clearly indicates that diabetes may influence malignant cell transformation by several mechanisms,including hyperinsulinemia,hyperglycemia and chronic inflammation.In this context,the receptor for advanced glycation end-products (RAGE) has emerged as a focal point in its contribution to malignant transformation and tumor growth.We high-light how RAGE,once activated,as it manifests itself in conditions such as diabetes or hyperglycemia,is able to continuously bring about an inflammatory milieu,thus supporting the contribution of chronic inflammation to the development of malignancies.     

Aminobisphosphonates Cause Osteoblast Apoptosis and Inhibit Bone Nodule Formation In Vitro

Bisphosphonates are widely used for the treatment of bone diseases associated with increased osteoclastic bone resorption. Bisphosphonates are known to inhibit biochemical markers of bone formation in vivo, but it is unclear to what extent this is a consequence of osteoclast inhibition or a direct inhibitory effect on cells of the osteoblast lineage. In order to investigate this issue, we studied the effects of various bisphosphonates on osteoblast growth and differentiation in vitro. The aminobisphosphonates pamidronate and alendronate inhibited osteoblast growth, caused osteoblast apoptosis, and inhibited protein prenylation in osteoblasts in a dose-dependent manner over the concentration range 20-100 microM. Further studies showed that alendronate in a dose of 0.1 mg/kg inhibited protein prenylation in calvarial osteoblasts in vivo, indicating that alendronate can be taken up by osteoblasts in sufficient amounts to inhibit protein prenylation at clinically relevant doses. Pamidronate and alendronate inhibited bone nodule formation at concentrations 10-fold lower than those required to inhibit osteoblast growth. These effects were not observed with non-nitrogen-containing bisphosphonates or with other inhibitors of protein prenylation and were only partially reversed by cotreatment with a fourfold molar excess of ss-glycerol phosphate. We conclude that aminobisphosphonates cause osteoblast apoptosis in vitro at micromolar concentrations and inhibit osteoblast differentiation at nanomolar concentrations by mechanisms that are independent of effects on protein prenylation and may be due in part to inhibition of mineralization. While these results need to be interpreted with caution because of uncertainty about the concentrations of bisphosphonates that osteoblasts are exposed to in vivo, our studies clearly demonstrate that bisphosphonates exert strong inhibitory effects on cells of the osteoblast lineage at similar concentrations to those that cause osteoclast inhibition. This raises the possibility that inhibition of bone formation by bisphosphonates may be due in part to a direct inhibitory effect on cells of the osteoblast lineage.