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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circulation. Author manuscript; available in PMC 2015 August 24.
Published in final edited form as:
PMCID: PMC4547604
NIHMSID: NIHMS713340

Response to Letter Regarding Article, “Proprotein Convertase Subtilisin Kexin Type 9 Promotes Intestinal Overproduction of Triglyceride-Rich Apolipoprotein B Lipoproteins Through Both Low-Density Lipoprotein Receptor–Dependent and –Independent Mechanisms”

In our Circulation article,1 we stated that few studies have addressed the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the small intestine and in the metabolism of triglyceride-rich lipoproteins. In their letter, Drs Le May and Cariou point out the omission of 2 references. The first, published by Le May et al2 in 2009, reports that PCSK9-/- mice display reduced postprandial hypertriglyceridemia and apolipoprotein B levels after olive oil gavage and that adenoviral PCSK9 overexpression or targeted shRNA silencing of PCSK9 increased and reduced apolipoprotein B secretion, respectively. In that article, Le May et al2 acknowledge an earlier observation, published by 1 of us (S.R.) in 2005, that apolipoprotein B48 levels are reduced in PCSK9−/− mice.3 This notwithstanding, we consider the article by Le May et al important and central to our story, and its omission was unintentional. The work was mentioned in an earlier draft, but the section was left out during manuscript rewriting to adjust the size to journal specifications. We apologize for the oversight and have prepared the correction notice that appears in this issue of the journal.

The second publication, by Levy et al4 in 2013, reports a role for PCSK9 in cholesterol absorption by showing that the addition of exogenous PCSK9 to CaCo-2 cell media affects NPC1L1 expression, a finding we also report in our Circulation article.1 However, we focused on mechanisms that directly account for increased intestinal apolipoprotein B production by PCSK9 and showed for the first time that mRNA levels of proteins (FAS, SCD, and DGAT2) involved in chylomicron assembly are increased by the addition of exogenous PCSK9 to CaCo-2 cells, that these effects are low-density lipoprotein receptor dependent, and that they are reversed by acute siRNA silencing of PCSK9 gene expression.1 Because we placed no emphasis on NPC1L1, we felt that not mentioning the article by Levy et al was appropriate given the space limitations. Le May and Cariou then ask whether in our study exogenous PCSK9 was added at the apical or basolateral side of CaCo-2 cells. We presented data from PCSK9 added to the apical side but obtained similar results after adding it to the basolateral side. This is in line with previous work in which Levy et al4 show that CaCo-2 cells secrete PCSK9 mostly into the basolateral medium, whereas Le May et al2 state that enterocytes show “strong PCSK9 immunostaining mainly in the apical compartment” and that “PCSK9…accumulates at the subapical and basolateral compartments of the enterocyte.”

Finally, Le May and Cariou question our claim of having used a physiological dose of PCSK9 in the studies of Caco-2 cells1 and suggest instead that the dose we chose is 2- to 3-fold higher than the highest physiological human PCSK9 concentration. We disagree with this statement because 10 μg/mL, the concentration we used to stimulate CaCo-2 cells, mimics the physiological effect of PCSK9 in vitro and is at the high end of the physiological PCSK9 concentration in humans.5

Footnotes

Disclosures: None.

Contributor Information

Shirya Rashid, Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, and Saint John, New Brunswick, Canada.

Hagai Tavori, Center of Preventive Cardiology, Knight Cardiovascular Institute, Oregon Health and Science University, Portland, OR.

Patrick E. Brown, Department of Biostatistics, Faculty of Medicine, University of Toronto and Cancer Care, Toronto, ON, Canada.

MacRae F. Linton, Department of Medicine, Section of Cardiovascular Disease Prevention, Vanderbilt University, Nashville, TN.

Jane He, Department of Medicine, Section of Cardiovascular Disease Prevention, Vanderbilt University, Nashville, TN.

Ilaria Giunzioni, Center of Preventive Cardiology, Knight Cardiovascular Institute, Oregon Health and Science University, Portland, OR.

Sergio Fazio, Center of Preventive Cardiology, Knight Cardiovascular Institute, Oregon Health and Science University, Portland, OR.

References

1. Rashid S, Tavori H, Brown PE, Linton MF, He J, Giunzioni I, Fazio S. Proprotein convertase subtilisin kexin type 9 promotes intestinal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-density lipoprotein receptor-dependent and -independent mechanisms. Circulation. 2014;130:431–441. doi: 10.1161/CIRCULATIONAHA.113.006720. [PMC free article] [PubMed] [Cross Ref]
2. Le May C, Kourimate S, Langhi C, Chétiveaux M, Jarry A, Comera C, Collet X, Kuipers F, Krempf M, Cariou B, Costet P. Proprotein convertase subtilisin kexin type 9 null mice are protected from postprandial triglyceridemia. Arterioscler Thromb Vasc Biol. 2009;29:684–690. doi: 10.1161/ATVBAHA.108.181586. [PubMed] [Cross Ref]
3. Rashid S, Curtis DE, Garuti R, Anderson NN, Bashmakov Y, Ho YK, Hammer RE, Moon YA, Horton JD. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc Natl Acad Sci U S A. 2005;102:5374–5379. doi: 10.1073/pnas.0501652102. [PubMed] [Cross Ref]
4. Levy E, Ben Djoudi Ouadda A, Spahis S, Sane AT, Garofalo C, Grenier É, Emonnot L, Yara S, Couture P, Beaulieu JF, Ménard D, Seidah NG, Elchebly M. PCSK9 plays a significant role in cholesterol homeostasis and lipid transport in intestinal epithelial cells. Atherosclerosis. 2013;227:297–306. doi: 10.1016/j.atherosclerosis.2013.01.023. [PubMed] [Cross Ref]
5. Lambert G, Ancellin N, Charlton F, Comas D, Pilot J, Keech A, Patel S, Sullivan DR, Cohn JS, Rye KA, Barter PJ. Plasma PCSK9 concentrations correlate with LDL and total cholesterol in diabetic patients and are decreased by fenofibrate treatment. Clin Chem. 2008;54:1038–1045. doi: 10.1373/clinchem.2007.099747. [PubMed] [Cross Ref]