Here we report that myeloid cells differentiating along the monocyte/macrophage lineage down-regulate the ST6Gal-I sialyltransferase via a protein kinase C/Ras/ERK signaling cascade. In consequence, the ␤1 integrin subunit becomes hyposialylated, which stimulates the ligand binding activity of ␣5␤1 fibronectin receptors. Pharmacologic inhibitors of protein kinase C, Ras, and MEK, but not phosphoinositide 3-kinase, block ST6Gal-I down-regulation, integrin hyposialylation, and fibronectin binding. In contrast, constitutively active MEK stimulates these same events, indicating that ERK is both a necessary and sufficient activator of hyposialylationdependent integrin activation. Consistent with the enhanced activity of hyposialylated cell surface integrins, purified ␣5␤1 receptors bind fibronectin more strongly upon enzymatic desialylation, an effect completely reversed by resialylation of these integrins with recombinant ST6Gal-I. Finally, we have mapped the N-glycosylation sites on the ␤1 integrin to better understand the potential effects of differential sialylation on integrin structure/function. Notably, there are three N-glycosylated sites within the ␤1 I-like domain, a region that plays a crucial role in ligand binding. Our collective results suggest that variant sialylation, induced by a specific signaling cascade, mediates the sustained increase in cell adhesiveness associated with monocytic differentiation.The U937 and THP-1 cell lines represent well accepted model systems for studying myeloid differentiation along the monocyte/macrophage lineage. Following treatment with phorbol myristate acetate (PMA), 4 these cells exhibit phenotypic changes that are characteristic of cell differentiation, including increased respiratory burst activity, enhanced phagocytotic capability, and markedly elevated cell adhesiveness to extracellular matrix ligands such as fibronectin. In vivo, the increased adhesiveness of monocytes/macrophages contributes to the extravasation of cells from the vasculature as well as tethering of cells within inflamed tissues.Differentiating myeloid cells bind to fibronectin through the integrin family of cell adhesion receptors, including the ␣5␤1 integrin species. The molecular mechanisms underlying PMA-dependent cell adhesion have not been well defined, although it has been reported that PMA increases the synthesis of both ␣5 and ␤1 integrin subunits (1-4). However, myeloid cells (U937 and THP-1) express an abundant amount of ␣5␤1 in the absence of PMA treatment, and yet these cells bind very poorly to fibronectin. This suggests that myeloid ␣5␤1 receptors are normally in an inactive state and that increased expression alone cannot account for the dramatically increased fibronectin binding induced by PMA.In our prior study (5) we observed that PMA stimulated a rapid but transient increase in fibronectin binding that was likely due to the activation of integrins already present on the cell surface. However, following this initial transient event there was a second phase of elevated fibronect...

Objective The objective of this study was to identify genetic variants associated with angiotensin-converting enzyme (ACE) inhibitor-associated angioedema. Participants and methods We carried out a genome-wide association study in 175 individuals with ACE inhibitor-associated angioedema and 489 ACE inhibitor-exposed controls from Nashville (Tennessee) and Marshfield (Wisconsin). We tested for replication in 19 cases and 57 controls who participated in Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial (ONTARGET). Results There were no genome-wide significant associations of any single-nucleotide polymorphism (SNP) with angioedema. Sixteen SNPs in African Americans and 41 SNPs in European Americans were associated moderately with angioedema (P<10−4) and evaluated for association in ONTARGET. The T allele of rs500766 in PRKCQ was associated with a reduced risk, whereas the G allele of rs2724635 in ETV6 was associated with an increased risk of ACE inhibitor-associated angioedema in the Nashville/Marshfield sample and ONTARGET. In a candidate gene analysis, rs989692 in the gene encoding neprilysin (MME), an enzyme that degrades bradykinin and substance P, was significantly associated with angioedema in ONTARGET and Nashville/Marshfield African Americans. Conclusion Unlike other serious adverse drug effects, ACE inhibitor-associated angioedema is not associated with a variant with a large effect size. Variants in MME and genes involved in immune regulation may be associated with ACE inhibitor-associated angioedema.

Background-Angioedema is a rare adverse effect of angiotensin converting enzyme (ACE) inhibitors that occurs more commonly in women and black Americans. Angioedema is thought to result from decreased degradation of vasoactive peptides. During ACE inhibition, bradykinin is primarily inactivated by aminopeptidase P (APP). Previous studies have provided conflicting data regarding serum APP activity in patients with a history of ACE inhibitor-associated angioedema. A single nucleotide polymorphism, −2399C>A (rs3788853, C-2399A), in XPNPEP2, the X-linked gene that encodes membranous APP, has been reported to associate with APP activity.

Differentiation of monocytes into macrophages is accompanied by increased cell adhesiveness, due in part to the activation of ␣4␤1 integrins. Here we report that the sustained ␣4␤1 activation associated with macrophage differentiation results from expression of ␤1 integrin subunits that lack ␣2-6-linked sialic acids, a carbohydrate modification added by the ST6Gal-I sialyltransferase. During differentiation of U937 monocytic cells and primary human CD14؉ monocytes, ST6Gal-I is down-regulated, leading to ␤1 hyposialylation and enhanced ␣4␤1-dependent VCAM-1 binding. Importantly, ST6Gal-I down-regulation results from cleavage by the BACE1 secretase, which we show is dramatically up-regulated during macrophage differentiation. BACE1 up-regulation, ST6Gal-I shedding, ␤1 hyposialylation, and ␣4␤1-dependent VCAM-1 binding are all temporally correlated and share the same signaling mechanism (protein kinase C/Ras/ ERK). Preventing ST6Gal-I down-regulation (and therefore integrin hyposialylation), through BACE1 inhibition or ST6Gal-I constitutive overexpression, eliminates VCAM-1 binding. Similarly, preventing integrin hyposialylation inhibits a differentiation-induced increase in the expression of an activation-dependent conformational epitope on the ␤1 subunit. Collectively, these results describe a novel mechanism for ␣4␤1 regulation and further suggest an unanticipated role for BACE1 in macrophage function.

Background The B2 receptor antagonist icatibant is approved for treatment of attacks of hereditary angioedema. Icatibant has been reported to decrease time-to-resolution of angiotensin-converting enzyme (ACE) inhibitor-associated angioedema in one study of European patients. Methods Patients with ACE inhibitor-associated angioedema (defined as swelling of lips, tongue, pharynx or face during ACE inhibitor use and no swelling in the absence of ACE inhibitor use) were enrolled at Vanderbilt University Medical Center from October 2007 through September 2015 and at Massachusetts General Hospital in 2012. C1 inhibitor deficiency and patients with bowel edema only were excluded. Patients were randomized within six hours of presentation to subcutaneous icatibant 30 mg or placebo at zero and six hours later. Patients assessed severity of swelling using a visual analog scale serially following study drug administration or until discharge. Results Thirty-three patients were randomized and 31 received treatment, with 13 receiving icatibant and 18 receiving placebo. One patient randomized to icatibant did not complete the visual analog scale and was excluded from analyses. Two-thirds of patients were African American and two-thirds were women. Time-to-resolution of symptoms was similar in placebo and icatibant treatment groups (p=0.19 for the primary symptom and p>0.16 for individual symptoms of face, lip, tongue, or eyelid swelling). Frequency of administration of H1 and H2 blockers, corticosteroids, and epinephrine was similar in the two treatment groups. Time-to-resolution of symptoms was similar in black and white patients. Conclusions This study does not support clinical efficacy of a bradykinin B2 receptor antagonist in ACE inhibitor-associated angioedema.