Evaluation of von Willebrand factor and von Willebrand factor propeptide in models of vascular endothelial cell activation, perturbation, and/or injury.

Pharmacologically, vasoactive agents targeting endothelial and/or smooth muscle cells (SMC) are known to cause acute drug-induced vascular injury (DIVI) and the resulting pathology is due to endothelial cell (EC) perturbation, activation, and/or injury. Alteration in EC structure and/or function may be a critical event in vascular injury and, therefore, evaluation of the circulatory kinetic profile and secretory pattern of EC-specific proteins such as VWF and VWFpp could serve as acute vascular injury biomarkers. In rat and dog models of DIVI, this profile was determined using pharmacologically diverse agents associated with functional stimulation/perturbation (DDAVP), pathological activation (lipopolysaccharide [LPS]/endotoxin), and structural damage (fenoldopam [FD], dopamine [DA], and potassium channel opener (PCO) ZD6169). In rats, FD caused moderate DIVI and time-related increase in plasma VWF levels ∼33% while in control rats VWF increased ∼5%. In dogs, VWF levels transiently increased ∼30% when there was morphologic evidence of DIVI by DA or ZD6169. However, in dogs, VWFpp increased >60-fold (LPS) and >6-fold (DDAVP), respectively. This was in comparison to smaller dynamic 1.38-fold (LPS) and 0.54-fold (DDAVP) increases seen in plasma VWF. Furthermore, DA was associated with a dose-dependent increase in plasma VWFpp. In summary, VWF and VWFpp can discriminate between physiological and pathological perturbation, activation, and injury to ECs.

An erythroid chaperone that facilitates folding of alpha-globin subunits for hemoglobin synthesis.

Erythrocyte precursors produce abundant alpha- and beta-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. alphaHb-stabilizing protein (AHSP) binds free alpha subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free alpha-globin caused by beta-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free alpha-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 alpha-globin genes (genotype Ahsp(-/-)alpha-globin*(alpha/alphaalpha)) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated alpha-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free alpha-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess alpha-globin, AHSP also acts as a molecular chaperone to stabilize nascent alpha-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances.

Loss of alpha-hemoglobin-stabilizing protein impairs erythropoiesis and exacerbates beta-thalassemia.

Hemoglobin (Hb) A production during red blood cell development is coordinated to minimize the deleterious effects of free alpha- and beta-Hb subunits, which are unstable and cytotoxic. The alpha-Hb-stabilizing protein (AHSP) is an erythroid protein that specifically binds alpha-Hb and prevents its precipitation in vitro, which suggests that it may function to limit free alpha-Hb toxicities in vivo. We investigated this possibility through gene ablation and biochemical studies. AHSP(-/-) erythrocytes contained hemoglobin precipitates and were short-lived. In hematopoietic tissues, erythroid precursors were elevated in number but exhibited increased apoptosis. Consistent with unstable alpha-Hb, AHSP(-/-) erythrocytes contained increased ROS and evidence of oxidative damage. Moreover, purified recombinant AHSP inhibited ROS production by alpha-Hb in solution. Finally, loss of AHSP worsened the phenotype of beta-thalassemia, a common inherited anemia characterized by excess free alpha-Hb. Together, the data support a model in which AHSP binds alpha-Hb transiently to stabilize its conformation and render it biochemically inert prior to Hb A assembly. This function is essential for normal erythropoiesis and, to a greater extent, in beta-thalassemia. Our findings raise the possibility that altered AHSP expression levels could modulate the severity of beta-thalassemia in humans.

Biomarkers and mechanisms of drug-induced vascular injury in non-rodents.

In preclinical safety studies, drug-induced vascular injury can negatively impact candidate-drug selection because there are no obvious diagnostic markers for monitoring this pathology preclinically or clinically. Furthermore, our current understanding of the pathogenesis of this lesion is limited. While vasodilatation and increased shear stress appear to play a role, the exact mechanism(s) of injury to the primary target cells, smooth muscle (SMC) and endothelial cell (EC), are unknown. Evaluation of potential novel markers for clinical monitoring with a mechanistic underpinning would add value in risk assessment and risk management. This mini review focuses on the efforts and progress to identify diagnostic markers as well as understanding the mechanism of action in nonrodent drug-induced vascular injury.