Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Apocynin-Treatment Reverses Hyperoxaluria Induced Changes in NADPH Oxidase System Expression in Rat Kidneys: A Transcriptional Study

<div><h3>Purpose</h3><p>We have previously shown that production of reactive oxygen species (ROS) is an important contributor to renal injury and inflammation following exposure to oxalate (Ox) or calcium-oxalate (CaOx) crystals. The present study was conducted, utilizing global transcriptome analyses, to determine the effect of Apocynin on changes in the NADPH oxidase system activated in kidneys of rats fed a diet leading to hyperoxaluria and CaOx crystal deposition.</p> <h3>Approach</h3><p>Age-, sex- and weight-matched rats were either fed regular rat chow or regular rat chow supplemented with 5% w/w hydroxy-L-proline (HLP). Half of the rats on the HLP diet were also placed on Apocynin-supplemented H₂O. After 28 days, each rat was euthanized, their kidneys freshly explanted and dissected to obtain both cortex and medulla tissues. Total RNA was extracted from each tissue and subjected to genomic microarrays to obtain global transcriptome data. KEGG was used to identify gene clusters with differentially expressed genes. Immunohistochemistry was used to confirm protein expressions of selected genes.</p> <h3>Results</h3><p>Genes encoding both membrane- and cytosolic-NADPH oxidase complex-associated proteins, together with <em>p21rac</em> and <em>Rap1a</em>, were coordinately up-regulated significantly in both renal medulla and cortex tissues in the HLP-fed rats compared to normal healthy untreated controls. Activation of NADPH oxidase appears to occur via the angiotensin-II/angiotensin-II receptor-2 pathway, although the DAG-PKC pathway of neutrophils may also contribute. Immuno histochemical staining confirmed up-regulated gene expressions. Simultaneously, genes encoding ROS scavenger proteins were down-regulated. HLP-fed rats receiving Apocynin had a complete reversal in the differential-expression of the NADPH oxidase system genes, despite showing similar levels of hyperoxaluria.</p> <h3>Conclusions</h3><p>A strong up-regulation of an oxidative/respiratory burst involving the NADPH oxidase system, activated via the angiotensin-II and most likely the DAG-PKC pathways, occurs in kidneys of hyperoxaluric rats. Apocynin treatment reversed this activation without affecting the levels of hyperoxaluria.</p> </div

Functional annotation chart showing differentially-expressed pathways in renal cortex tissue comparing HLP+Apocynin treated versus untreated control groups.

<p>The gene heading indicates number of genes mapped to an ontology category. The first ten pathways are common between the cortex and the medulla. P-values derived from Fisher's exact test and Benjamini multiple test correlation.</p

Summary of the proposed activation of Nox-2 and Nox-4 NADPH oxidases in the kidney.

<p>The figure shows various cytosolic and membrane components of NADPH oxidase subunits getting activated via Renin/angiotensin receptor-2 activation of the NADPH-oxidases during the development of hyperoxaluria, resulting in increased production of reactive oxygen species (ROS).</p

Immuno histochemical staining of paraffin embedded kidney sections of HLP treated rats.

<p>(<b>A</b>) Osteopontin (Opn) associated with crystal deposits, Single arrows show staining of crystal deposits while double arrows point to staining of epithelial cells of the adjoining renal tubular epithelia. (<b>B</b>) Double arrows point to strong staining for the monocyte chemoattractant protein (Mcp-1) in renal tubular epithelial cells (<b>C</b>) Staining for Nox-4 in tubular epithelial cells (<b>D</b>) Staining for Nox-2 in both tubular cells as well as interstitium and (<b>E</b>) general cytosolic staining for p47 in tubular epithelial cells.</p

Relative expressions of genes in HLP-fed rats vs. control compared to HLP+ Apocynin fed rats vs. control.

<p>(<b>A</b>) Genes encoding the membrane components of NADPH oxidase such as gp91^Phox or Nox2, p22^Phox, and Rap1a as well as cytosolic components such as p47, p40 and p67. gp91^Phox is substituted by Nox4 in the distal tubular cells of the renal cortex of the NADPH oxidase complex (<b>B</b>) Genes encoding the oxygen scavengers such as catalase (cat) and superoxide dismutase (sod1) are shown to be down regulated in the HLP-fed rats (Red bars) and up regulated in the HLP-Apocynin fed rats (Green bars). Most of the genes depicted had values >0.3 (or >2 fold ratio differential-expression).</p

Representative Pizzolato staining of paraffin embedded kidney sections of Hydroxy-L-Proline (HLP) treated rats.

<p>(<b>A</b>) Light micrograph section showing little calcium oxalate crystal depositions shown with single arrows at 10X. (<b>B</b>) Light micrograph section of the kidney showing brownish-black stained calcium oxalate crystal depositions shown with single arrows with the oral administration of 5% hydroxy-l-proline (10X). Majority of the crystals were seen in the tubular lumens of the distal tubules and collecting ducts of the cortex and outer medulla. There were no crystal deposits in normal rat kidneys.</p

Relative expressions of selected genes encoding proteins in the HLP-fed rats and the HLP+Apocynin fed rats.

<p>As expected the HLP-fed rats had osteopontin (<i>Opn</i>), monocyte chemoattractant protein (<i>Mcp1</i>) and matrix gla protein (<i>Mgp</i>) to be up regulated and Tamm-horsefall protein (<i>Thp</i>) to be down regulated. The results were reversed with the HLP+Apocynin treated group. Most of the genes depicted had values >0.3 (or >2 fold ratio differential-expression).</p

Relative expressions of genes in HLP-fed rats vs. control compared to HLP+ Apocynin fed rats vs. control.

<p>Genes downstream of angiotensin receptor-2 encoding important immune cell-associated signaling pathways for the activation of NADPH oxidase expressed in the cortex or medulla of HLP-treated rats showing up regulation (Red bars), but exhibiting down-regulation in the cortex and medulla of HLP+Apocynin-treated rats (Green bars).</p