# Patent

## Title
LRP1 as key receptor for the transfer of sterified cholesterol from very-low-density lipoproteins (VLDL) to ischaemic cardiac muscle

## Abstract

The invention relates to novel molecules that can modulate one of the mechanisms leading to the massive deposition of cholesterol in the cardiomyocytes and/or in the smooth muscle cells of the vascular wall, during acute myocardial infarction or other clinical situations involving ischaemia. The invention also shows that the blockage of LRP1 by means of certain agents, including a recombinant expression vector, an RNAi, an antibody, a siRNA etc., prevents the overaccumulation of esterified cholesterol in the cardiomyocytes and/or in the smooth muscle cells of the vascular wall exposed to ischaemia. The invention also relates to the use of said molecules in the treatment and/or prevention of the changes in the metabolism of calcium and cardiac remodeling associated with ischaemia.

## Background
The invention is of interest to the pharmaceutical and chemical sectors and refers to new molecules capable of modulating the mass deposition of cholesterol in cardiomyocytes so that said molecules have clinical application, as they have therapeutic effects by minimizing cardiac alterations of acute myocardial infarction or angina pectoris induced by dyslipidemia or other conditions that occur alongside ischaemia.
BACKGROUND
During ischaemic processes related to acute myocardial infarction or angina pectoris, a lipid accumulation in the myocardium is produced. This fact has not only been observed in test animals (Straeter-Knowlen I M et al, Circulation 1996; Chabowski A et al, FEBS Lett 2006) but also in patients (Golfbarb J W et al, Radiology 2009). Furthermore, there is also experimental evidence showing that dyslipidemia contributes to the exacerbation of cardiac alterations induced by ischaemia in animal models (Osipov R M et al, Circulation 2009; Kim E et al, J Neurosci 2008). It has recently been demonstrated that high doses of VLDL alter calcium (Ca2+) regulation in cardiomyocytes and that these alterations induced by VLDL are exacerbated in situations of hypoxia, with SERCA-2 sarcoplasmic reticulum protein playing a crucial role (Castellano J et al, J Mol Cell Cardiol 2011).
Ischaemia as a basis of heart failure results in the condition known as ischaemic cardiomyopathy. Ischaemic cardiomyopathy is with high frequency a result of coronary disease, whose underlying pathology is atherosclerosis (Gersh B J et al, journal 1997). Atherosclerotic plaque evolution produces a progressive imbalance between oxygen supply and demand in the myocardium. The gravest outcome of the atherosclerotic process, infarction, is in 80% of cases due to atherosclerotic plaque rupture, the formation of the thrombus, and the total or partial occlusion of the vessel (Burke A P et al, Med Clin North America 2007). Dyslipidemia is a key risk factor in ischaemic cardiomyopathy generation, mainly because of its role at the beginning and in the development of atherosclerosis. In situations of dyslipidemia there is an increase in the influx of lipoproteins towards the arterial intimae, where they are modified by means of oxidation and aggregation through interaction with the proteoglycanes that make up the extracellular matrix (Sartipy P et al, Circ Res 2000; Hakala J K et al, ATVB 2001). Modified lipoprotein uptake by smooth muscular cells from the vascular wall and macrophages leads to the formation of foam cells in the vascular wall. Low-density lipoprotein receptor-related protein (LRP1) has been identified as a key receptor for uptake of LDL modified by intracellular cholesterol aggregation and accumulation in smooth muscle cells from the vascular wall and macrophages as well as for their transformation into foam cells (Llorente-Cortés V et al, ATVB 2000; Llorente-Cortés et al, ATVB 2002; Llorente-Cortés et al, J Lipid Res 2007, Llorente-Cortés et al, Cardiovasc Res 2007). LRP1 is upregulated in advanced atherosclerotic lesions rich in lipids (Luoma J et al, J Clin Invest 1994; Llorente-Cortés et al, Eur J Clin Invest 2004) and may be considered as a heart disease biomarker as there are clinical trials showing the relationship between LRP1 expression alteration and coronary disease (Handschug K et al, J Mol Med 1998; Schulz et al, Int J Cardiol 2003). It has also been shown that risk factors relevant for atherosclerosis development such as hypercholesterolemia and hypertension upwards regulate LRP1 expression in the vascular wall (Llorente-Cortés et al, Circulation 2002; Sendra J et al, Cardiovasc Res 2008). It has later been shown that aggregated LDL uptake by LRP1 regulates the expression and activation of the tissue factor, a main coagulation activator, therefore regulating thrombus formation by means of a RhoA and sphyngomyelin-dependant mechanism (Llorente-Cortés et al, Circulation 2004; Camino-López S et al, Cardiovasc Res 2007; Camino-López S et al, J Thromb Haemost 2009). In addition to all these processes it has been described how LRP1 takes part in the regulation of extracellular matrix composition (Strickland D K et al, Trends Endocrinol Metab 2002), may promote receptor internalization (Wu L et al, J Cell Biochem, 2005) and regulates the activity of various intracellular signaling proteins (Herz J et al, J Clin Invest 2001).
The role of LRP1 in cardiomyocytes and the consequences of alterations in its expression for lipid metabolism are wholly unknown at this time. It is known that lipase lipoprotein present in the surface of cardiomyocytes mediates LDL uptake. Nonetheless, lipase lipoprotein uses a receptor so far unidentified for the selective uptake of cholesterol, and it is not the classic LDL receptor (Yagyu H et al, J Clin Invest 2003; Yokoyama M et al, J Lipid Res 2007). It has also been demonstrated that LRP1 mediates selective cholesterol uptake in vascular cells (Llorente-Cortés et al, ATVB 2006).
Cardiomyocytes accumulate lipids in different pathophysiological conditions, of which processes of acute ischaemia are one (Chabowski A et al, FEBS Lett 2006; Goldfarb J W et al, Radiology 2009), though the mechanisms by which this occurs are to a large extent unknown. One of the mechanisms participating in triglyceride (TG) accumulation in ischaemic situations is the increase of endogenous TG synthesis due to hypoxia (Boström P et al, ATVB 2006). Nonetheless, cardiomyocytes may catch lipids from lipoproteins rich in TG such as VLDL and chylomicrons. It is known that CD36 and lipase lipoprotein participate in the uptake of VLDL fatty acids by cardiomyocytes (Bharadwaj K G et al, J Biol Chem 2010). As CD36 is increased in situations of hypoxia (Mwaikambo B R et al, J Biol Chem 2009), it is plausible that increased CD36 levels participate in the accumulation of TG observed in the ischaemic heart. Although it is known that the heart may take up cholesterol from VLDL (Bharadawaj K G et al, J Biol Chem 2010) and chylomicrons (Fielding C J et al, J Clin Invest 1978), the mechanisms involved in the entry of cholesterol into the heart are completely unknown.
Ischaemia is associated with an electromechanical dysfunction and with severe alterations in intracellular calcium dynamics (García-Dorado D et al, Cardiovasc Res 2006; Takukder M A et al, Cardiovasc Res 2009), key for cardiac functionality. Moreover, it recently has been demonstrated that cardiomyocytes exposed to high doses of VLDL suffer from intracellular lipid accumulation, a decrease in SERCA-2 expression, calcium amplitude and sarcoplasmic reticulum calcium content. These effects were exacerbated by exposing the cultivated myocytes to a hypoxic environment. The results highlight the central role of SERCA-2 in the empowerment by hypoxia of alterations in calcium handling induced by VLDL (Castellano J et al, JMCC 2011). It has also recently been described how LRP1 is upregulated by hypoxia in smooth muscular cells from the vascular wall (Castellano J et al, ATVB 2011). These data demonstrate the importance of understanding the molecular mechanisms involved in lipid uptake by cardiomyocytes in ischaemic situations and the importance of knowing the role of LRP1 in this process.
Therefore, there is currently a need to develop a method for modulating lipoprotein receptors such as LRP1 to treat and prevent the accumulation of neutral lipids such as cholesteryl ester (CE) in cardiomyocytes during ischaemic events, which would minimize the cardiac alterations of acute myocardial infarction or angina pectoris, induced by dyslipidemia or other conditions that accompany ischaemia.
BRIEF DESCRIPTION OF THE INVENTION
The present invention refers to the identification of a key receptor for the entry of cholesterol into the heart in ischaemic situations, LRP1. It also refers to the design of molecules such as lentiviruses, antibodies or transcription factors capable of modulating the activity of this receptor and therefore preventing cardiac alterations associated with the entry of neutral lipids or cholesterol into cardiomyocytes exposed to acute ischaemia processes such as acute myocardial infarction or angina pectoris or to chronic ischaemia processes.
DETAILED DESCRIPTION OF THE INVENTION
At present, the mechanisms leading to cholesterol accumulation in cardiomyocytes are completely unknown. The present invention demonstrates that in ischaemic situations upwards regulation of the receptor LRP1 in the cardiomyocyte. LRP1 overexpression in ischaemic situations plays a primary role in the transfer of cholesteryl ester from lipoproteins into the cardiomyocyte as well as in the intracellular accumulation of this lipid in the heart. Furthermore, the present invention demonstrates that the modulation of the expression and/or function of LRP1 prevents cholesteryl ester overaccumulation in cardiomyocytes exposed to ischaemia. Therefore, this invention provides new molecules capable of modulating one of the mechanisms leading to the massive deposition of cholesterol in the cardiomyocyte during acute myocardium infarction or other conditions that accompany ischaemia.
In the present invention “acute myocardial infarction” refers to an insufficient blood supply, with tissue damage, in a portion of the heart caused by a blockage in one of the coronary arteries, frequently due to the rupture of a vulnerable atheroma plaque. Ischaemia, hypoxia or deficient oxygen supply resulting from such obstruction produces angina pectoris, which does not produce cardiac tissue death if it is precociously rechanneled, whereas if this anoxia is sustained, myocardial lesioning and eventually necrosis occur, which is to say, infarction.
In the present invention it is demonstrated that in cardiomyocytes exposed to hypoxia, overexpression of the receptor LRP1 occurs in association with an increase in the intracellular levels of one of the neutral lipids, cholesteryl ester. It is also demonstrated that modulation, preferably blockage, of LRP1 overexpression induced by hypoxia makes it possible to slow and/or inhibit (partially or fully) the entry of cholesteryl ester from lipoproteins into the heart.
In the present invention it is demonstrated that in the hearts of patients with ischaemic cardiomyopathy there is a very high expression of receptor LRP1, and that expression of this receptor correlates very significantly with the cholesteryl ester content of the myocardium. This does not occur in patients with idiopathic dilated cardiomyopathy or control subjects. Therefore, this mechanism is specific and is only relevant in ischaemic cardiomyopathy.
In the present invention, low density lipoprotein receptor-related Protein is known as “LRP1”, also known as α2-macroglobulin, CD91 or AI316852. Said receptor is codified by gene Mm. 271854 in Homo sapiens. 
In the present invention the term “ischaemia” or “ischaemic cardiomyopathy (ICM)” refers to a set of alterations in heart functionality present when arteries supplying blood and oxygen to the heart are blocked. There is generally an accumulation of cholesterol and other substances called plaque in the arteries that supply oxygen to myocardial tissue. Over time, the myocardium does not work well and it is more difficult for the heart to fill up and to pump blood. Ischaemia is a common cause of cardiac congestive insufficiency. Patients with this condition may have had a heart attack, angina or unstable angina at some time and it is possible that some patients may not have noticed any previous symptoms. In these patients lipids have been found in the penumbra zone (periphery of the area of infarction), though the mechanisms involved in these accumulations are unknown at this time. What is known is that these lipids are involved in inducing severe alterations in calcium metabolism and cardiac remodeling, keys to cardiac functionality.
In the present invention idiopathic dilated cardiomyopathy refers to cardiac muscle illnesses in which the structure and function of the myocardium are altered in absence of coronary illness, hypertension, valvulopathies, or congenital heart defects which explain said anomalies. Among the various myocardiopathies, dilated myocardiopathy (DMC) is defined by the presence of dilatation and systolic dysfunction affecting the left ventricle or both ventricles.
The present invention also refers to a method for avoiding cholesteryl ester accumulation in cardiomyocytes, characterized in that the pharmaceutical composition comprises at least one LRP1 expression and/or function modulator agent. Preferably said expression and/or function modulator agent is selected from the following group: lentivirus, antibody and transcription factor. Said agent is preferably selected from the group below:
a) agents which modulate the expression of LRP1, on a transcriptional level, such as transcription factors (HIF-1alpha (see example 8), SREBPs or others), specific siRNAs released to cells, or activating or inhibitory molecules of the degradation pathway of LRP1 (see example 9); and/or
b) agents which modulate the function of LRP1, such as molecules capable of specifically inhibiting the cholesterol uptake function of LRP1.
In the present invention the term “transcription factors” defined in a) refers to those factors that are able to respectively increase or decrease LRP1 expression.
In the present invention the term “specific siRNAs released to cells” defined in a) refers to those siRNAs for inhibiting LRP1 expression that can be released to the cells by lentiviruses and by other means such as electroporation, which are widely known in the state of the art.
In the present invention the term “activating or inhibitory molecules of the degradation pathway of LRP1” defined in a) refers to those molecules described in the ubiquitin-proteasome system.
In the present invention the term “molecules capable of specifically inhibiting the cholesterol uptake function of LRP1” defined in b) refers to those molecules, preferably proteins, which are able to compete with LRP1 in the binding of lipoprotein ligands, such as specific LRP1—monoclonal or polyclonal- or peptides, more concretely, peptides isolated from aminoacids sequence cluster II from the alpha chain of LRP1 protein (SEQ. ID No: 19). In a more preferable embodiment of the invention, said molecules prevent against the pathophysiological function of LRP1 without altering its essential functions.
In the present invention is described an agent that modulates the expression and/or function of the protein LRP1 is described, which is a protein which competes with LRP1 in binding lipoprotein ligands, more precisely it is a specific antibody from an aminoacid sequence or a peptide comprised in cluster II of the alpha chain of the protein LRP1 (SEQ. ID No: 19), and more precisely a specific polyclonal antibody from an aminoacid sequence or peptide which belongs to the following group (see Examples 10 and 11):

    
    
        peptide P1 (SEQ ID No: 13),
        peptide P2 (SEQ ID No: 14), and
        peptide P3 (SEQ ID No: 15).
    
    


Those skilled in the art of biotechnology and molecular biology and with the information described within the present invention may obtain a specific monoclonal antibody from an aminoacid sequence or peptide comprised in cluster II of the alpha chain of the protein LRP1 (SEQ ID No: 19) and more precisely a specific monoclonal antibody from an aminoacid sequence or peptide which belongs to the following group: peptide P1 (SEQ ID No 13), peptide P2 (SEQ ID No: 14) and peptide P3 (SEQ ID No: 15). Therefore, another object of the invention is constituted by a monoclonal antibody that modulates the expression and/or function of the protein LRP1 that is specific to a sequence of aminoacids or peptide comprised in cluster II of the alpha chain of the protein LRP1 (SEQ ID No: 19), or more precisely specific to a peptide which belongs to the following group:

    
    
        peptide P1 (SEQ ID No: 13),
        peptide P2 (SEQ ID No: 14), and
        peptide P3 (SEQ ID No: 15).
    
    


Furthermore, the peptides described in the present invention, which are useful for obtaining an antibody—whether polyclonal or monoclonal—that modulates the function of protein LRP1 which may be obtained from an aminoacid sequence or a peptide comprised in cluster II of the alpha chain of the protein LRP1 (SEQ ID No: 19), and more precisely the peptides P1 (SEQ ID No: 13), P2 (SEQ ID No: 14) and P3 (SEQ ID No: 15), constitute another particular object of the present invention.
The present invention also refers to a pharmaceutical composition characterized in that it comprises at least one LRP1 expression modulator agent and/or one LRP1 function modulator agent defined above to control the expression of lipoprotein receptors such as VLDLR, LDLR or LRP1 during acute myocardial infarction or other clinical conditions which accompany ischaemia, hypoxia or deficient oxygen supply, which pharmaceutical composition is preferably characterized in that it comprises an expression modulator agent and/or an LRP1 function modulator agent defined above for blocking LRP1 expression during ischaemia.
The present invention also refers to the use of a pharmaceutical composition defined above to regulate the accumulation of neutral lipids such as cholesteryl ester, triglycerides or free cholesterol in cardiomyocytes of a patient with an acute myocardial infarction or other clinical condition which accompanies ischaemia, hypoxia or deficient oxygen supply such as cardiac insufficiency, ischaemic cardiomyopathy or idiopathic dilated cardiomyopathy.
The present invention refers to a method for regulating the accumulation of neutral lipids such as cholesteryl ester, triglycerides or free cholesterol accumulation in cardiomyocytes, characterized by administering to a patient a therapeutically effective amount of a pharmaceutical composition to control the expression of lipoprotein receptors such as VLDLR, LDLR or LRP1 during an acute myocardial infarction or other clinical condition which accompanies ischaemia, hypoxia or deficient oxygen supply such as cardiac insufficiency, ischaemic card iomyopathy or idiopathic dilated card iomyopathy.
The present invention also refers to a method for avoiding cholesteryl ester accumulation in cardiomyocytes, characterized by administering to the patient a therapeutically effective amount of a pharmaceutical composition to block LRP1 expression during ischaemia, the patient preferably suffering from ischaemic cardiomyopathy.
A preferred embodiment of the invention refers to the use of said pharmaceutical composition defined above to avoid, slow, partially or totally inhibit cholesteryl ester accumulation in cardiomyocytes of a patient suffering or who has suffered from ischaemic cardiomyopathy.
The present invention also protects a method for the treatment and/or prevention of cardiac alterations associated with the entry of neutral lipids or cholesterol into cardiomyocytes exposed to acute ischaemia processes such as acute myocardial infarction or angina pectoris, or to chronic ischaemia.
Finally, the present invention protects the use of molecules capable of modulating lipoprotein receptors such as VLDLR, LDLR or LRP1 in the manufacturing of a medicine used to prevent and/or treat cardiac alterations associated with mass neutral lipid deposition in cardiomyocytes during acute myocardium infarction or other clinical situations which accompany ischaemia processes.
Throughout the description and the claims, the word “comprises” and its variants is not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and characteristics of the invention shall be inferred in part from the description and practice of the invention. The following figures and examples are given by way of a non-limiting illustration of the present invention.



DESCRIPTION OF THE DRAWINGS
FIG. 1. The effect of hypoxia on LRP1, VLDLR and LDLR expression in cardiomyocytes. A) The quiescent HL-1 cardiomyocytes were exposed to normoxia or hypoxia during increasing times and gene expression of LRP1, VLDLR and LDLR was analyzed by means of real-time PCR. Data were processed with a software program based on the relative calculation of mRNA concentration according to the Ct (threshold cycle) value. Data were normalized by the endogenous control ARBP and were expressed as the mean±SEM of three experiments carried out in duplicate. *P<0.05 vs. cardiomyocytes in normoxia. B) Western blot analysis showing LRP1, VLDLR and LDLR bands in both HL-1 and NRVM exposed to normoxia or hypoxia for 18-24 hours. β-tubulin and GAPDH levels were displayed as a protein load control for HL-1 and NRVM cells, respectively.
FIG. 2. The effect of VLDL on LRP1, VLDLR and LDLR expression and on neutral lipid (free cholesterol, cholesteryl ester and triglycerides) accumulation in HL-1 exposed to normoxia or hypoxia.
The quiescent cardiomyocytes were exposed to VLDL under conditions of normoxia or hypoxia. A) Analysis by real-time PCR of changes in LRP1, VLDLR and LDLR expression induced by increasing doses of VLDL. Data were processed with a software program based on the relative calculation of mRNA concentration according to the Ct (threshold cycle) value. Data were normalized by the endogenous control ARBP. B) A Western blot is displayed representing LRP1, VLDLR and LDLR protein expression levels in HL-1 exposed to VLDL (1.8 mM, 18 hours) in normoxia or hypoxia. β-tubulin levels are displayed as a protein load control. C) Thin layer chromatography showing the bands of cholesteryl ester (CE), triglycerides (TG) and free cholesterol (FC) and a line graph with quantification of these bands in HL-1 cells exposed to normoxia (dotted lines) or hypoxia (continuous lines). Results were shown as micrograms of CE or TG per milligram of cellular protein and were expressed as the mean±SEM of three experiments carried out in duplicate. *P<0.05 vs. cardiomyocytes without VLDL: #P<0.05 vs. cardiomyocytes in normoxia.
FIG. 3. Design of the miR RNAi sequences and study of their efficiency in blocking LRP1 expression in HL-1 and NRVM. Analysis of LRP1 mRNA expression in HL-1 cells infected with RNAi1919, RNAi8223 or RNAi8531. Data were processed with a software program based on the relative calculation of mRNA concentration according to the Ct (threshold cycle) value. Data were normalized by the endogenous control ARBP. The results were expressed as the percentage of expression with respect to cells infected with the negative control. *P<0.05 vs. cells transfected with the negative control.
FIG. 4. Use of the BLOCK-ITTM RNAi Designer system to inhibit LRP1 expression (XM—001056970) by means of its incorporation into lentiviruses used to infect HL-1 and NRVM. (A) Generation of the miR RNAi expression vector by annealing the oligos and ligation into the pcDNATM6.2_GW/miR linear vector. (B) Transfer of miR RNAi constructions to pDONRTM221 vectors by means of the reaction catalyzed by BP clonase II. (C) Transfer of the miRNA sequence from the clone pENTRTM221 along with pENTR5′-CMV plasmid to vector 0.4/R4R2/V5-DESTTM by means of the reaction catalyzed by LR clonase II.
FIG. 5. The effect of RNAi8531 on LRP1 expression and on the accumulation of CE and TG originating from VLDL in HL-1 exposed to normoxia or hypoxia. HL-1 stably transfected with RNAi8531 or with the negative control were cultivated for 48 hours in the presence of blasticidin. The cells were subjected to quiescence for 24 hours and were later exposed to normoxia (N, grey bars) or hypoxia (H, black bars) A) A Western blot representative of LRP1, VLDLR and LDLR expression in HL-1 infected with the negative control or with miR8531 as well as quantification of the corresponding bands is displayed. β-tubulin levels are displayed as a protein load control. B) The quiescent HL-1 cells were exposed to normoxia or hypoxia for 24 hours and during the last 12 hours were incubated with VLDL (1.8 mM). A representative thin layer chromatography is displayed with the cholesteryl ester (CE), triglyceride (TG) and free cholesterol (FC) bands and the histograms with the quantification of the CE and TG bands. The results were expressed as micrograms of lipids per milligram of protein and were displayed as the mean±SEM of three experiments carried out in triplicate. *P<0.05 vs. cells infected with the negative control. C) The quiescent HL-1 cells were exposed to normoxia or hypoxia for 24 hours and during the last 12 hours were incubated with doubly radiolabeled VLDLs (1.8 mM). The cells were collected and cholesterol and triglyceride uptake was evaluated by means of a dpm count of [3H] and [14C] associated with the cell extracts, respectively. The results were expressed as [3H] or [14C] dpm per milligram of cellular protein. [3H]/[14C] cellular ratio was also determined. The results were expressed as the mean±SEM of three experiments carried out in triplicate. *P<0.05 vs. HL-1 in normoxia. #P<0.05 vs. cardiomyocytes infected with the negative control.
FIG. 6. The effect of RNAi8531 on the expression of LRP1 and on the accumulation of CE and TG originating from VLDL in NRVM exposed to normoxia or hypoxia. NRVM transiently transfected with RNAi8531 or with the negative control were cultivated for 48 hours in the presence of blasticidin. The cells were subjected to quiescence for 24 hours and were later exposed to normoxia (N, grey bars) or hypoxia (H, black bars) A) A Western blot representative of the expression of LRP1, VLDLR and LDLR in NRVM infected with the negative control or with miR8531 as well as the quantification of the corresponding bands. GAPDH levels are displayed as a protein load control. B) The quiescent NRVM cells were exposed to normoxia or hypoxia for 24 hours and during the last 12 hours were incubated with VLDL (1.8 mM). A representative thin-layer chromatography is displayed with the cholesteryl ester (CE), triglyceride (TG) and free cholesterol (FC) bands, and the histograms with the quantification of the CE and TG bands. The results were expressed as micrograms of lipids per milligram of protein and were displayed as the mean±SEM of three experiments carried out in triplicate. *P<0.05 vs. cells infected with the negative control.
FIG. 7. The effect of ischaemia induced by acute infarction on neutral lipid accumulation and lipoprotein receptor expression in the myocardium (porcine model). Samples from a non-ischaemic zone (remote, white bars) and ischaemic (penumbra, black bars) were taken from the hearts of pigs subjected to infarction (n=6) or controls (n=3, sham) and were frozen in liquid nitrogen. One part of each sample was used for lipid extraction and thin layer chromatography, and the other part to obtain protein for the Western blot. A) Western blot analysis displaying the protein expression levels of LRP1 and VLDLR and bar graph showing the quantification of the bands. The levels of protein expression of GAPDH are displayed as a protein load control B) Thin layer chromatography displaying the bands of cholesteryl ester (CE), triglycerides (TG) and free cholesterol (FC), and histograms displaying the quantification of the CE and TG bands. Data were expressed as the mean±SEM. P<0.05 vs. non-ischaemic myocardium or sham animals.
FIG. 8. Analysis of the expression of LRP1 in hearts explanted from patients with idiopathic cardiomyopathy (DCM) or with ischaemic cardiomyopathy (ICM) as compared to control subjects (CNT). A) A Western blot analysis of LRP1 protein expression was performed using the protein obtained from 50 mg of myocardial tissue homogenized in TriPure reagent. GAPH levels were used as a load control. B) Representative image of LRP1 immunohistochemical analysis in myocardial tissue. Magnification×240.
FIG. 9. Analysis of VLDLR expression in hearts explanted from patients with idiopathic cardiomyopathy (DCM) or with ischaemic cardiomyopathy (ICM) as compared to control subjects (CNT). A) A Western blot analysis of VLDLR protein expression was performed using the protein obtained from 50 mg of myocardial tissue homogenized in TriPure reagent. GAPH levels were used as a load control. B) Representative image of VLDLR immunohistochemical analysis in myocardial tissue. Magnification×240.
FIG. 10. Determination of myocardial levels of cholesteryl ester (CE), triglycerides (TG) and free cholesterol (FC) in hearts explanted from patients with idiopathic dilated cardiomyopathy (DCM) or with ischaemic cardiomyopathy (ICM) as compared to control subjects (CNT). Lipid extraction and subsequent thin-layer chromatography were performed using 5 mg of myocardial tissue (A) in order to determine the levels of cholesteryl ester (CE) (B), triglycerides (TG) (C), and free cholesterol (FC) (D) CNT, controls; DCM, idiopathic dilated cardiomyopathy; ICM, ischaemic cardiomyopathy. The results were expressed as micrograms per milligram of tissue and were displayed as mean±SD.
FIG. 11. Correlation between LRP1 expression and cholesteryl ester (CE) content in the myocardium of patients with cardiac insufficiency. Analysis of the correlation between mRNA expression of LRP1 (A) or protein expression of LRP1 (B) and CE content of the myocardium.
FIG. 12. Correlation between VLDLR expression and cholesteryl ester (CE) content in the myocardium of patients with cardiac insufficiency. Analysis of the correlation between the mRNA expression of VLDLR (A) or the protein expression of VLDLR (B) and the CE content of the myocardium.
FIG. 13. Modulation of the expression of the receptor LRP1 by transcription factor HIF-1α in cardiomyocytes. The cardiomyocytes from the line HL-1 were transfected with siRNA-anti-HIF-1α or siRNA-random (0.6 μmol/L) by nucleofection. Control cells were nucleofected in the absence of siRNA. The cells were exposed to normoxia or hypoxia and were collected after 4 hours to analyze the expression of HIF-1α and after 24 hours to analyze the expression of LRP1. A) A representative Western blot image and bar graph displaying HIF-1α and LRP1 quantification. β-tubulin levels are displayed as a load control. The results were expressed as the mean±SEM of two experiments carried out in triplicate. *P<0.05 vs. HL-1 in normoxia. B) Quantification by real-time PCR of LRP1, VLDLR and LDLR gene expression in HL-1 exposed to normoxia and hypoxia for 24 hours. Data were processed with a software program based on the relative calculation of mRNA concentration according to the Ct (threshold cycle) value. Data were normalized by the endogenous control ARBP and were expressed as the mean±SEM of three experiments carried out in duplicate. *P<0.05 vs. HL-1 in normoxia; # vs. control cells.
FIG. 14. The protein E3 ubiquitin ligase CHFR modulates the stability of the protein LRP1 in smooth muscle cells from the human vascular wall (VSMC). The VSMC were transfected with siRNA-random or siRNA-anti-CHFR and were later exposed to low density lipoproteins modified by aggregation (agLDL). A) Bar graphs displaying the quantification by real-time PCR of mRNA expression levels for CHFR. Data were processed with a software program based on the relative calculation of mRNA concentration according to the Ct (Threshold cycle) value. Data were normalized by the endogenous control GAPDH and were expressed as the mean±SEM of three experiments carried out in triplicate. B) A representative Western blot image of the protein expression levels and a bar graph displaying the quantification of LRP1 (C) and CHFR (D) bands normalized by the levels of β-tubulin. *P<0.05 vs. cells not exposed to agLDL. #P<0.05 vs. VSMC treated with siRNA-random.
FIG. 15. Low-density lipoproteins modified by aggregation (agLDL) stabilize the protein LRP1 by means of a decrease in CHFR expression and ubiquitination of the cytoplasmic chain in smooth muscle cells from the human vascular wall (VSMC). A representative Western blot image of the protein expression of CHFR (A) and LRP1 (B) and a bar graph displaying the quantification of the respective bands normalizing the results with β-tubulin. (C) The control VSMC or the VSMC exposed to agLDL (100 μg/mL, 24 hours), were incubated with cycloheximide (100 μM) for the indicated times. β-tubulin levels were displayed as a load control. Line graph showing the percentage of protein expression after adding cycloheximide. The results are displayed as the mean±SEM of two independent experiments carried out in triplicate. (D) The VSMC protein extracts exposed to agLDL were applied over affinity beads with anti-ubiquitin antibodies for the immunoprecipitation of ubiquitinated proteins. A fraction of the cellular extracts was applied over balls bound to unspecific antibodies. Later, the ubiquitinated LRP1 protein was analyzed by Western blot. The “Westerns” were incubated with anti-ubiquitin antibodies demonstrating that ubiquitin expression was not altered by agLDL treatment. *P<0.05 vs. cells not exposed to agLDL.
FIG. 16. Effect of the polyclonal antibodies on intracellular accumulation of lipids from LDL modified by aggregation (agLDL) in smooth muscular cells from the human vascular wall (VSMC). The quiescent VSMC cells were exposed to hypoxia for 18 hours. Polyclonal antibodies (anti-P1, anti-P2 and anti-P3) (100 μg/mL) were later added to the culture medium. After 3 hours aggregated LDL (100 μg/mL) were added to the same culture medium and were kept for 4 more hours. The cells were collected in NaOH, and the lipid extraction and thin-layer chromatography were performed. A representative thin-layer chromatography is displayed showing the cholesteryl ester (CE) and free cholesterol (FC) bands and histograms with the quantification of the CE bands. The results were expressed as micrograms of lipid per milligram of protein and were displayed as the mean±SEM of three experiments carried out in triplicate. *P<0.05 vs. control cells.
FIG. 17. Effect of the polyclonal antibodies on intracellular accumulation of lipids from VLDL in HL-1 cardiomyocytes exposed to hypoxia. The quiescent HL-1 cells were exposed to hypoxia for 24 hours. Polyclonal antibodies (anti-P1, anti-P2 and anti-P3) (100 μg/mL) were later added to the culture medium, and after 4 hours VLDL (75 μg/mL) were added to the same culture medium and were kept for 4 hours. HL-1 cardiomyocytes were collected in NaOH, after which lipid extraction was performed followed by a thin layer-chromatography. A representative thin layer chromatography with the cholesteryl ester (CE), triglyceride (TG) and free cholesterol (FC) bands and the histograms with the quantification of the CE and TG bands are displayed. The results were expressed as micrograms of lipid per milligram of protein and were displayed as the mean±SEM of three experiments carried out in triplicate. *P<0.05 vs. control cells.



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EXAMPLES
The following specific examples provided in this patent are intended to illustrate the nature of the present invention. These examples are included for illustrative purposes only and are not to be interpreted as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting the field of application thereof.
Example 1
Effect of Hypoxia on the Expression of Lipoprotein Receptors by the Cardiomyocyte
Adult HI-1 cardiomyocytes or neonatal rat cardiomyocytes (NRVM) were exposed to normoxia or hypoxia conditions. The expression of LRP1, VLDL receptor (VLDLR) and classic LDL receptor (LDLR) was analyzed at mRNA level by means of real-time PCR and at protein level by means of a Western blot analysis. (FIG. 1)
It was observed that in cardiomyocytes subjected to normoxia, the expression levels of LRP1 and VLDLR were very low, whereas levels of LDLR were very high, especially in adult HL-1 cardiomyocytes. This expression pattern was completely altered by exposing the cardiomyocytes to hypoxia. As observed in FIG. 1A, hypoxia induced in a time-dependant way the expression of LRP1 (from 1.6-fold after 4 hours to 2.7-fold after 16 hours) and also the expression of VLDLR (from 2.8-fold after 4 hours to 4-fold after 16 hours), while classic LDL receptor expression decreased (from 10% after 4 hours to 34% after 16 hours). After 24 hours the expression of receptors LRP1 and VLDLR was significantly increased, while the expression of LDLR was decreased by hypoxia both in HL-1 and NRVM (Table 1). As was also observed through PCR, the result of the Western blot demonstrated that hypoxia increased LRP1 and VLDLR expression while decreasing the expression of the classic LDL receptor in both types of cells studied (FIG. 1B).







TABLE 1







Effect of hypoxia on the gene expression of lipoprotein receptors in


cardiomyocytes










HL-1
NRVM












Normoxia
Hypoxia
Normoxia
Hypoxia















LRP1
0.8 ± 0.2
 3.5 ± 0.2*
1.2 ± 0.2
3.1 ± 0.9*


VLDLR
3.2 ± 0.5
15 ± 2*
 0.6 ± 0.07
 1.8 ± 0.34*


LDLR
225 ± 31 
91 ± 4*
0.8 ± 0.2
0.4 ± 0.1*





HL-1 and NRVM cardiomyocytes were exposed to normoxia or hypoxia for 24 hours. LRP1, mRNA expression of VLDLR and LDLR was determined by real-time PCR. Data were processed by means of a program based on the Ct value and were normalized by the expression of the endogenous ARBP control. Data were expressed as the mean ± SEM of three experiments carried out in duplicate.


*P < 0.05 vs. cells exposed to conditions of normoxia.






Example 2
Effect of VLDL on the Expression of Lipoprotein Receptors (LRP1, VLDLR and LDLR) and on the Accumulation of Neutral Lipids (CE, TG and FC) 1N HL-1 Exposed to Normoxia or Hypoxia
Cells exposed to VLDL in normoxia or hypoxia were collected and the study of the expression of lipoprotein receptors and the determination of intracellular lipids was performed. The results in FIG. 2A demonstrated that VLDL in every dose tested inhibited the expression or mRNA of LDLR in normoxic or hypoxic cells, and that the highest dose of VLDL increased the expression of LRP1 in hypoxic HL-1. Expression of VLDLR was not significantly altered by the presence of VLDL. The Western blot analysis (FIG. 2B) confirmed at protein level the results obtained at mRNA level. The thin-layer chromatography assays (FIG. 2C) demonstrated that VLDL induced intracellular lipid accumulation in a VLDL-dose dependant way from 6.7±2.2 to 77.6±8.8 μg CE/mg of cellular protein and from 2.5±0.2 to 69.17±3.0 μg TG/mg of cellular protein. Hypoxia also significantly increased said accumulation at every VLDL dose tested (i.e. at 3.6 mM up to 110.52±1.6 μg CE/mg of cellular protein or up to 80.54±3.85 μg TG/mg of cellular protein)
In order to analyze the viability of the cultures, we analyzed the cellular levels of a survival marker (Bcl2) and an apoptosis marker (BAX). Neither VLDL nor hypoxia altered BAX levels. Nonetheless, there was a reduction of Bcl2 levels from 5.39±0.35 to 4.48±0.18 by VLDL (1.8 mM, 18 hours) up to 3.32±0.13 by hypoxia (18 hours) and up to 2.85±0.01 by VLDL (1.8 mM) and hypoxia (18 hours) together. These results demonstrate that our experimental conditions do not induce apoptosis but reduce the survival capacity of the HL-1 cells in culture.







TABLE 2







Effect of VLDL and hypoxia on HL-1 apoptosis










VLDL
Bcl2
BAX
Bcl2/BAX













(mM)
Normoxia
Hypoxia
Normoxia
Hypoxia
Normoxia
Hypoxia
















0
5.4 ± 0.4 
3.3 ± 0.1#
10.4 ± 0.1
11.3 ± 0.6#
0.5
0.3


1.8
5.0 ± 0.20
3.3 ± 0.2#
 9.4 ± 0.5
10.9 ± 0.4#
0.5
0.3


3.6
4.5 ± 0.2*
 2.9 ± 0.1*#
11.1 ± 0.6
 12.2 ± 0.4*#
0.4
0.2





HL-1 cardiomyocytes were exposed to increasing doses of VLDL under conditions of normoxia or hypoxia for 16 hours. The gene expression of Bcl2, BAx and CPP32 was determined by means of real-time PCR. Data were processed by a program based on Ct value and were normalized by the endogenous control ARBP. Data were displayed as the mean ± SEM of four experiments carried out in duplicate.


*P < 0.05 vs. cells incubated in the absence of VLDL;


#P < 0.05 vs. cells exposed to conditions of normoxia.






Example 3
Design of a Lentivirus to Inhibit Expression of the Receptor LRP1
A) Different miR RNAi sequences were designed using the Invitrogen BLOCK-ITTM RNAi Designer to inhibit expression of LRP1 (XM—001056970) by incorporating it into lentiviruses used to infect HL-1 and NRVM: the sequences SEQ ID No: 1, and SEQ ID No: 2 were designed to inhibit the expression of LRP1 (miR RNAi-XM—001056970—1919 or RNAi1919 (SEQ ID No: 16)); the sequences SEQ ID No 3: and SEQ ID No: 4 were designed to inhibit the expression of LRP1 (miR RNAi-XM—001056970—8223 or RNAi8223 (SEQ ID No: 17)); and the sequences SEQ ID No: 5 and SEQ ID No: 6 were designed to inhibit the expression of LRP1 (miR RNAi-XM—001056970—8531 or RNAi8531 (SEQ ID No: 18)).
The plasmid pcDNATM 6.2-GW/miR was used as a negative control in all the transfection experiments: SEQ ID No: 7 and SEQ ID No: 8 sequences were designed for universal negative control (pcDNATM 6.2-GW/miR-neg).
B) Analysis of expression of mRNA of LRP1 in HL-1 cells infected with RNAi1919 (SEQ ID No: 16), RNAi8223 (SEQ ID No: 17) or RNAi8531 (SEQ ID No: 18) (FIG. 3) Data were processed by a software program based on the calculation of relative mRNA according to the Ct (threshold cycle) value. Data were normalized by the endogenous control ARBP. Data were expressed as the percentage of expression with respect to the cells infected with the negative control. *P<0.05 vs. transfected cells with negative control. Artificially synthesized SEQ ID No: 9 was also used as vector pLenti6.4TM-CMV-MSGW.
BLOCK-ITTM RNAi Designer is a web tool which makes it possible to design and order specific siRNA or miRNA oligonucleotides against concrete target sequences. After entering the target sequence into the program and setting a series of restrictive parameters, the program generates several RNAi designs which are ordered according to their probable efficiency in inhibiting the expression of the target gene. In this case, the program has generated three sequences capable of inhibiting the LRP1 gene [RNAi1919 (SEQ ID No: 16), RNAi8223 (SEQ ID No: 17) or RNAi8531 (SEQ ID No:18)] as well as the negative control (pcDNATMGW/miR_neg). The pcDNATM-GW/miR_neg plasmid contains an insert forming a “hairpin” structure which is processed in the form of a mature miRNA unable to bond to any known gene in vertebrates. Therefore, this plasmid may be used as a negative control for the knockdown experiments performed with the expression vectors pcDNATM6.2-GW/miR. In order to generate the expression vector miR RNAi the oligos were annealed and ligated to the linear vector pcDNATM6.2-GW/miR (SEQ ID No 10) (FIG. 4A). Next, competent E. Coli cells were transformed with the ligated DNA and the cells were selected in LB medium supplemented with 50 μg/mL of spectinomycin. The miR RNAi constructions were later transferred to the pDONRTM221 (SEQ ID No: 11) by means of a reaction catalyzed by BP clonase II (FIG. 4B). The miRNA sequence was then transferred from the pENTRTM221 (SEQ ID No: 12) clone along with the pENTR5′-CMV (Invitrogen) plasmid to the vector pLenti6.4/R4R2/V5-DESTTM (SEQ ID No: 23) by means of a reaction catalyzed by LR clonase II (FIG. 4C). Competent E. Coli cells were transformed with the recombinant expression vector and the cells were selected in LB supplemented with ampicillin.
In the present invention, the term “recombinant expression vector” refers to a small plasmid containing a multiple cloning site flanked by one or two promoter sequences, such as pLenti6.4TM-CMV-V5-MSGW/miR (Life Technologies (Invitrogen)). These promoters are required in order to transcribe the DNA fragments inserted in the multiple cloning site. In this case, the inserted DNA sequence refers to the RNAi sequences previously described.
The colonies were picked and cultured overnight in LB medium with ampicillin. Digestion with Hind was performed to verify that recombination had taken place correctly. The lentivirus (pLenti6.4TM-CMV-MSGW/miR-XM-001056970-1919 (SEQ ID No: 21); pLenti6.4TM-CMV-MSGW/miR-XM-001056970-8223 (SEQ ID No: 24); pLenti6.4TM-CMV-MSGW/miR-XM-001056970-8531 (SEQ ID No: 20); pLenti6.4TM-CMV-MSGW/miR-neg (Life Technologies (Invitrogen)) were used to transfect the cardiomyocytes. The ability of these sequences to inhibit the mRNA expression of LRP1 was determined by real-time PCR. It was observed that pLenti6.4TM-CMV-MSGW/miR-XM-001056970-1919 (SEQ ID No: 21) and pLenti6.4TM-CMV-MSGW/miR-XM-001056970-8531 (SEQ ID No: 20) were able to inhibit the mRNA expression of LRP1 in hypoxic cardiomyocytes in about 50% (FIG. 3). pLenti6.4TM-CMV-MSGW/miR-XM-001056970-8531 (SEQ ID No: 20) was chosen to stably transfect HL-1 and transiently infect NRVM. Another example of how to inhibit LRP1 expression in the cardiomyocyte could be the use of floxed recombinant alleles and recombination by Cre-resulting in the deletion of a part of the LRP1 promoter including the transcription starting site.
Other examples of how to inhibit the expression of LRP1 could be by modulating the nuclear levels of transcription factors with the ability to modulate the expression of LRP1. Specifically, our group has published that the transcription activity LRP1 is negatively modulated by SREBP transcription factors (Llorente-Cortés V et al, Circulation 2002; Llorente-Cortés V et al, J Mol Biol 2006; Costales P et al, Atherosclerosis 2010), and positively by HIF-1α (Castellano J et al, Arterioscler Thromb Vasc Biol 2011) in smooth muscle cells from the vascular wall. Therefore, it is highly probable that SREBP overexpression and HIF-1α inhibition may be strategies worth taking into account in order to inhibit the upwards regulation of LRP1 by hypoxia in cardiomyocytes. Apart from these strategies focused on preventing the upwards regulation of LRP1 induced by hypoxia, it might be more appealing, from a pharmacological point of view, to inhibit the ability of LRP1 to transfer cholesterol from atherogenic lipoproteins to cardiomyocytes in dyslipidemic situations, as this has a lower degree of impact on the physiological functioning of LRP1. To do this, it is certainly essential to develop molecules capable of impeding or hindering the ability of LRP1 to transfer cholesterol to the cardiomyocyte when an ischemic situation occurs, moreover if the patient is dyslipidemic. To this end, we have stably transfected COS cells capable of secreting mini receptors of LRP1, molecules that could be effective in competing with cellular LRP1 competing for the binding of lipoproteins.
Example 4
Effect of LRP1 Cellular Deficiency on the Transference of Cholesteryl Ester and Triglycerides from VLDL to Cardiomyocytes Exposed to Normoxia or Hypoxia
RNAi8531 (SEQ ID No: 18) reduced the protein expression of LRP1 by 50.7±1.14% in HL-1 (FIG. 5A) and by 40.6±1.8% (FIG. 6A) in NRVM with respect to the same cells transfected with the negative control. Nonetheless, RNAi8531 (SEQ ID No: 18) had no effect over the low protein expression of LRP1 in normoxia. No differences were found in the protein expression of VLDLR or LDLR among the cells transfected with negative control or RNAi8531 (SEQ ID No: 18) independently of whether they were exposed to normoxia or hypoxia. Therefore, with this method, we obtained cardiomyocytes specifically deficient in LRP1 receptor expression. We then studied the effect of VLDL on the intracellular accumulation of CE and TG in LRP1-deficient cardiomyocytes exposed to normoxia and hypoxia with respect to the control variety. Our results demonstrated that overaccumulation of CE induced by hypoxia in HL-1 (FIG. 5B) and NRVM (FIG. 6B) was completely prevented in LRP1-deficient cells. Nonetheless, intracellular overaccumulation of TG induced by hypoxia was similar in LRP1-deficient cells and control cells. These results suggest that the LRP1 deficiency specifically affected CE uptake from VLDL, and that therefore the cholesterol and triglyceride uptake pathways may be independent. In order to study CE and TG uptake specifically, the cardiomyocytes were exposed to VLDL in which the cholesterol and triglyceride components were marked with two different radioisotopes. CE from VLDL was marked with [3H] and TG with [14C]. The specific activity of [3H] was of 439±54 dpm/mg of protein and that of [14C] was of 87±15 dpm/mg of protein. Our results demonstrated that LRP1 deficiency completely prevented the increase in the uptake of CE (2.3-fold) induced by hypoxia (FIG. 5C). Nonetheless, the increase in TG uptake inducted by hypoxia (1.3-fold) was similar in both control and LRP1-deficient cells (FIG. 5C). The ratio [3H]/[14C] was 5±0.8 in the double-marked VLDL, whereas in the HL-1 cells this ratio was 1.04±0.19, indicating that [3H] uptake by HL-1 cells in normoxia is very low. Hypoxia increased two-fold the uptake of [3H] in control cells, but not in LRP1-deficient cells. Therefore, these results demonstrate that LRP1 exerts a crucial role in selective cholesteryl ester uptake by cardiomyocytes exposed to hypoxia, and that blocking this receptor has a clear impact on cholesteryl ester accumulation in cardiomyocytes. Given that VLDL is one of the heart's main sources of cholesterol, LRP1 blockage may be a key to reducing the supply of cholesterol supply to the ischaemic heart without altering the supply of triglycerides.
Example 5
Effect of Ischaemia Inducted by Acute Infarction on Lipid Accumulation in the Heart
An experimental model of acute myocardial infarction was used in the previously described porcine model (Vilahur G et al, J Moll Cell Cardiol 2011; Vilahur G et al, J Thromb Haemost 2009). We evaluated the effect of hypoxia on the accumulation of neutral lipids (CE, TG and FC), and on the expression of lipoprotein receptors (LRP1 and VLDLR) in the non-ischaemic zone (remote myocardium zone) and in the ishacemic zone (penumbra or perinecrotic zone). The lipid pattern was determined by the lipid extraction from the samples and the subsequent thin-layer chromatography. It was demonstrated that cholesteryl ester levels and triglyceride levels increased 2- and 3-fold, respectively, in the ischaemic myocardium vs. the non-ischaemic or control myocardium (FIG. 7A). There was no change in the free cholesterol level between the ischaemic myocardium and the non-ischaemic or control myocardium.
Expression levels of the different lipoprotein receptors were also determined by a Western blot analysis. It was observed that LRP1 protein expression increased 5.7-fold in the ischaemic myocardium vs. the non-ischaemic or control myocardium, P<0.05. An increase in VLDLR protein expression was also observed in the ischaemic zone, although a much more moderate one (2-fold) (FIG. 7B). These results demonstrate that in an acute ischaemic process such as the one induced by acute myocardial infarction, there is a significant increase in LRP1 expression in the penumbra zone of the myocardium, concomitantly with an increase of neutral lipids content.
Example 6
Analysis of Lipoproteic Receptor Expression in Myocardium Samples from Control Subjects, Patients with Idiopathic Dilated Cardiomyopathy or with Ischaemic Cardiomyopathy
A total of 55 explanted human hearts were chosen from 26 patients with dilated idiopathic cardiomyopathy (DCM) and from 29 patients with ischaemic cardiomyopathy (ICM) (Table 3). Hearts from 4 healthy people who had died in traffic accidents, and whose hearts could not be used for transplant were used as controls.







TABLE 3







Characteristics of the patients according to the etiology of the


cardiac insufficiency











dilated (DCM)
ischaemic (ICM)




(n = 26)
(n = 29)
P














Cardiomyopathy





Age (years)
56 ± 3
54 ± 3
0.566


Gender
86/14
93/1


(men/women)


(%)


NYHA (%)
Class III = 79
Class III = 47



Class IV = 21
Class IV = 53


Cholesterol plasma
 4.49 ± 0.29
 3.89 ± 0.28
0.833


(mmol/L)


PWTd (mm)
73.60 ± 5.43
67.60 ± 2.84
0.043


EF (%)
20.20 ± 2.66
25.00 ± 2.48
0.779


Coronariography


1 vessel (%)
7
27


2 vessels (%)
0
 7


3 vessels (%)
0
53









The results were expressed as the mean±SD for continuous variables and as a percentage of patients in the categorical variables. PWTd: posterior wall thickness of the left ventricle in diastole. EF: ejection fraction. DCM: dilated cardiomyopathy; ICM: ischaemic cardiomyopathy.

The myocardial sample (25 mg) obtained from explanted hearts was ground and homogenized in TriPure isolation reagent. The gene and proteic expression of lipoproteic receptors LRP1, VLDLR and LDLR was determined by means of real-time PCR and Western blot analysis, respectively.







TABLE 4







Lipoprotein receptor gene expression in the myocardium in cardiac


insufficiency, by etiology.











Control
DCM
ICM



(n = 4)
(n = 26)
(n = 29)














LRP1
9.53 ± 3.14
7.65 ± 3.45
12.33 ± 11.53*#


VLDLR
28.85 ± 9.66 
22.01 ± 12.04
27.30 ± 14.92


LDLR
11.14 ± 10.90
 3.19 ± 1.94*
 3.75 ± 4.57*









The frozen myocardial tissue (25 mg) was ground and homogenized in the TriPure isolation reagent. LRP1, VLDLR and LDLR gene expression was determined by real-time PCR. Data were processed by means of a software program based on the Ct value and were normalized by the expression of the endogenous 18srRNA control. Controls; DCM: dilated cardiomyopathy; ICM: ischaemic cardiomyopathy. Data were expressed as the mean±SD. *P<0.05 vs. controls. #P<0.05 vs. DCM.

Compared to expression in DCM and control hearts, hearts from ICM patients showed increased levels of LRP1 both at mRNA level (12.33±11.53 vs. 7.65±3.45 or 9.53±3.14, P<0.05) (Table 4) and protein level (22.27±20.66 vs. 8.64±7.22 or 6.11±0.74, P<0.05) (FIG. 8A). These results were also corroborated by immunohistochemistry (FIG. 8B). On the contrary, VLDLR expression at protein level was increased by cardiomyopathy, independently of its etiology (ICM: 15.48±10.61 and DCM: 11.64±4.90 vs. CNT: 3.68±2.98, P=0.05 and P=0.01, respectively) (FIG. 9A). Immunohistochemical results confirmed the absence of differences in VLDLR expression between myocardium of DCM and ICM patients (FIG. 9B). LDL receptor decreased in cardiomyopathic situations independently of its etiology (ICM: 3.75±4.57 and DCM: 3.19±1.94 vs. 11.14±10.90, P=0.016 y P=0.012, respectively) (Table 4).
Example 7
Analysis of Neutral Lipid Levels in Myocardium Samples from Control Subjects, Patients with Idiopathic Dilated Cardiomyopathy (DCM) or with Ischaemic Cardiomyopathy (ICM)
The thin-layer chromatography results (FIG. 10A) demonstrated that the myocardium from patients with ICM had higher levels of CE than those of patients with DCM or controls (92.2±68.3 vs. 49.7±9.9 or 39.0±1.8, P=0.05) (FIG. 10B). Although the TG level was higher in the myocardium from patients with DCM or ICM with respect to the controls (111.3±60.7 and 100.2±44.4 vs. 50.8±39.4, P=0.05 and P=0.04) (FIG. 10C), no significant differences were observed among these two groups of patients. There were no differences in free cholesterol content in hearts from the different groups studied (FIG. 10D). It was found that LRP1 myocardial expression correlated very significantly with CE content both at mRNA level (R2=0.69, P<0.0001) (FIG. 11A) and at protein level (R2=0.66, P<0.0001) (FIG. 11B). In a lower grade than with LRP1, VLDLR myocardial expression also positively correlated with CE content at mRNA level (R2=0.32, P=0.03) (FIG. 12A) and at protein level (R2=0.27, P<0.01) (FIG. 12B). The correlation between LRP1 or VLDLR and cholesteryl ester content in the heart was specifically observed in the ischaemic cardiomyopathy group (Table 5).







TABLE 5







Correlations between lipoprotein receptor expression and


cholesteryl ester content in myocardium, by etiology of the cardiac


insufficiency.












DCM (n = 10)

ICM (n = 13)













R2
P
R2
P

















LRP1 mRNA
0.012
0.075
0.74
<0.0001



LRP1 protein
0.032
0.65
0.78
<0.0001



VLDLR mRNA
0.001
0.93
0.27
0.05



VLDLR protein
0.031
0.63
0.33
0.05







DCM: dilated cardiomyopathy,



ICM: ischaemic cardiomyopathy.






Example 8
Hypoxia does not Increase LRP1 Expression in HIF-1α-Deficient Cardiomyocytes
To analyze the role of HIF-1α in the upregulatory effect of hypoxia on LRP1 expression, HIF-1α was inhibited with a specific siRNA (Applied Biosystems, siRNA ID 4390815) by a nucleofection technique. siRNA-anti HIF1α, though not the unspecific siRNA-random, completely prevented the increase in HIF-1α (3.8-fold) induced by hypoxia (FIG. 13A, left panel) in HL-1 cardiomyocytes. Hypoxia does not exert any significant effect on LRP1 expression in HIF-1α-deficient HL-1 cardiomyocytes (FIG. 13A, right panel). In contrast, hypoxia significantly increases LRP1 expression in control and siRNA-random transfected HL-1 cardiomyocytes. As shown in FIG. 13B, real-time PCR experiments show that HIF-1α inhibition efficiently prevents the upregulatory effect of hypoxia on LRP1 and VLDLR expression. However, HIF-1α inhibition does not exert any significant effect on the downregulatory effect of hypoxia on the classical receptor for LDL (LDLR).
Example 9
E3 CHFR Ubiquitin Ligase Protein Modulates LRP1 Protein Stability and Mediates the Effect of Aggregated LDL in the Ubiquitination of the LRP1 Beta Chain
In order to discover the effect of the protein CHFR on LRP1 protein levels, CHFR mRNA expression was silenced though nucleofection by specific siRNA. As shown in FIG. 14A, both siRNA-CHFR (Applied Biosystems, siRNA ID s 31393) and aggregated LDL (agLDL) (100 μg/mL) decreased CHFR mRNA expression by 70% and 50%, respectively. The simultaneous exposure of cells to siRNA-CHFR and agLDL reduces CHFR mRNA expression up to 30%. In line with these results, Western blot experiments show that CHFR protein levels were reduced by 56% with agLDL, by 39% with siRNA-CHFR and by 27% by both interventions applied together (FIG. 14B & FIG. 14C). Concomitantly with CHFR reduction, LRP1 expression was increased by agLDL (1.72-fold), by siRNA-CHFR (1.9-fold) and by joint interventions (2.26-fold) (FIG. 14B & FIG. 14D). As shown in FIG. 15A, agLDL (100 μg/mL, 20 hours) reduced CHFR protein expression by 33% and increased LRP1 protein expression 2.5-fold. CHFR decrease induced by agLDL leads to a reduction in LRP1 beta chain ubiquitination (FIG. 15B) and to an increase in LRP1 protein half-life (FIG. 15C).
Example 10
Anti-P2 and Anti-P3 Antibodies Reduce the Intracellular Accumulation of Cholesteryl Ester Induced by Aggregated LDL Internalization in Human Vascular Smooth Muscle Cells Exposed to Hypoxic Conditions
As shown in FIG. 16, anti-P2 and anti-P3 antibodies were able to efficiently compete with aggregated LDL (agLDL) for LRP1 binding. Therefore, anti-P2 and anti-P3 antibodies efficiently and significantly inhibit intracellular cholesteryl ester accumulation derived from agLDL uptake. Anti-P2 and anti-P3 antibodies decrease intracellular cholesteryl ester accumulation by 50% and 30%, respectively, in hypoxic human vascular smooth muscle cells (VSMC).
Example 11
Anti-P1 Antibodies Reduce the Intracellular Accumulation of Cholesteryl Ester Originating from the Uptake of VLDL in Hypoxic Cardiomyocytes
As shown in FIG. 17, anti-P1 antibodies were able to efficiently compete with VLDL and to significantly inhibit intracellular cholesteryl ester accumulation originating from VLDL uptake by 56% in hypoxic HL-1 cardiomyocytes.
Example 12
Milestone I. Obtaining Monoclonal Antibodies and Small-Size Molecules for Application in In Vivo Models of Acute Myocardial Infarction
On the basis of polyclonal antibodies obtained following the procedure described in this report, monoclonal antibodies and small size molecules are obtained, which are able to inhibit the uptake of cholesteryl ester from VLDL by cardiomyocytes in in vivo models.
To carry out this task, we have worked in collaboration with the Antibody Production Service of the Universidad Autönoma de Barcelona (director: Antoni Iborra). Antibody purification and endotoxin removal are also carried out, so as to be able administer the antibodies in the in vivo model.
Milestone II. Role of the Inhibition of the Receptor LRP1 in Cardioprotection in In Vivo Models of Acute Myocardial Infarction
The effect of the molecules developed in milestone I are studied in:
IIa. Cardiomyocyte Cell Cultures.
IIb. In Vivo Models of Acute Myocardial Infarction: Mouse and Pig.
Milestone II takes place in the Cardiovascular Research Center, CSIC-ICCC, which is equipped with laboratories, equipment and specialized scientific researchers in order to successfully carry out the following experimental procedures: cell cultures, animal facility, genomics platform, proteomics laboratory, confocal microscopy with resonance scanner, immunohistochemistry and immunocytochemistry platforms, and flow cytometry platform.
*Each one of these platforms is run by a postdoctoral researcher. The center has the platforms and methodology required to carry out most of the studies. Most of the technology required is already up and running and has been adapted to suit the objectives set out by this project.
Example 13
Materials and Methods
13.1 Cell Cultures
I. HL-1 Cell Line Cultures
The HL-1 cell line was kindly provided by Dr. W. C. Claycomb (Louisiana State University Medical Centre, New Orleans, La., USA). These cells show similar characteristics to those of cardiomyocytes. These cells were kept in Claycomb medium (JRH Biosciences, Leneka, Kans., USA) supplemented with 10% of foetal calf serum (Invitrogen Corporation, Carlsbad, Calif., USA), norepinephrine (100 μM), penicillin (100 U/mL), streptomycin (100 μg/mL) and amphotericin (0.25 μg/mL) in cell culture dishes covered with fibronectin (12.5 μg/mL) and gelatine (0.02%) at 37° C. and at 5% CO2.
II. Isolation and Cell Culture of Neonatal Rat Ventricular Myocytes (NRVM)
This study was approved by the Animal Research Committee of the Instituto Catalan de Ciencias Cardiovasculares (ICCC020/DMAH4711) and was carried out according to the Guide for the Care and Use of Experimental Animals published by the U.S. National Institute of Health. The NRVM cells were prepared from the ventricles of hearts of rats 3-4 days old. The neonates were killed by decapitation, their hearts were extracted and Atria separated. Homogeneous solutions were prepared using the kit “Neonatal Cardiomyocyte Isolation System” (Worthington Biochemical Corporation), following the manufacturer's instructions. Cardiomyocytes were separated from cardiac fibroblasts by pre-seeding these in plastic wells at 37° C. and 5% CO2 for 90 min. The cardiomyocytes were maintained in wells covered with 1% gelatin in DMEM:M199 (Gibco) supplemented with 5% FBS, 10% horse serum (Invitrogen) and 1% P/S. 1 μg/mL of cytosine b-D-arabinofuranoside (Sigma) was added to the culture medium to inhibit residual proliferation of fibroblasts. The culture medium was changed every other day. After 48 hours of culture, myocytes exhibited regular spontaneous contraction. Cells were used for experiments after 3-6 days in culture.
III. Generation of LRP1-Deficient Cardiomyocytes
Design of miRNA Lentivirus to Inhibit LRP1 Expression
A) Different miR RNAi sequences were designed using the Invitrogen BLOCK-ITTM RNAi Designer system to inhibit LRP1 expression (XM—001056970) by incorporation into lentiviruses used to infect HL-1 and NRVM:

    
    
        the sequences SEQ ID No: 1 and SEQ ID No: 2 correspond to the top and bottom oligonucleotides of miR RNAi designed and artificially synthesized to inhibit LRP1 expression (miR RNAi-XM—001056970—1919 or RNAi1919);
        the sequences SEQ ID No: 3 and SEQ ID No: 4 correspond to the top and bottom oligonucleotides of miR RNAi designed and artificially synthesized to inhibit LRP1 expression (miR RNAi-XM—001056970—8223 or RNAi8223);
        the sequences SEQ ID No: 5 and SEQ ID No: 6 correspond to the top and bottom oligonucleotides of miR RNAi designed to inhibit LRP1 expression (miR RNAi-XM—001056970—8531 or RNAi8531);
    
    


The plasmid pcDNATM 6.2-GW/miR was used as a negative control in all transfection experiments: SEQ ID No: 7 and SEQ ID No: 8 correspond to the top and bottom oligonucleotides of the universal negative control (pcDNATM6.2-GW/miR-neg).
B) Analysis of LRP1 mRNA expression in HL-1 cells infected with RNAi1919 (SEQ ID No: 16), RNAi8223 (SEQ ID No: 17) or RNAi8531 (SEQ ID No: 18) (FIG. 3). The data were processed with a software program based on the relative calculation of mRNA concentration according to the Ct value (threshold cycle). The data were normalized by the endogenous control ARBP. The results were expressed as the percentage of expression compared to the cells infected with the negative control. *P<0.05 vs. cells transfected with the negative control.
In our case, the program has generated three sequences with the ability to inhibit the gene LRP1 [RNAi1919 (SEQ ID No: 16), RNAi8223 (SEQ ID No: 17) or RNAi8531 (SEQ ID No: 18)] in addition to the negative control (pcDNATM-GW/miR_neg). The pcDNATM-GW/miR_neg plasmid contains an insert which forms a hairpin structure that is processed in a mature miRNA that is unable to bind to any known genes in vertebrates. Therefore, this plasmid serves as a negative control for the knockdown experiments carried out with pcDNATM6.2-GW/miR expression vectors. The artificially-synthesized sequences SEQ ID No: 9 and SEQ ID No: 10, which correspond to sequences of DNA of vectors pLenti6.4TM-CMV-MSGW and pcDNATM6.2_GW, respectively, were also used.
IV. Production of the Lentivirus Particles
In order to generate the miR RNAi expression vector, oligos were annealed and ligated into the linear vector pcDNATM6.2_GW/miR (SEQ ID No: 10). Subsequently, competent E. coli cells were transformed with the ligated DNA and cells were selected in LB medium supplemented with 50 μg/mL of spectinomycin. miR RNAi constructs were then transferred to pDONRTM221 (SEQ ID No: 11) vectors through the reaction catalyzed by the BP clonase II (FIG. 4B).
The sequence miRNA of the clone pENTRTM221 (SEQ ID No: 12) was then transferred together with the pENTR5′-CMV plasmid (Invitrogen) to the pLenti6.4/R4R2/V5-DESTTM vector (SEQ ID No: 23) using the reaction catalyzed by LR clonase II (FIG. 4C). The artificially synthesized sequences SEQ ID No: 11 and SEQ ID No: 12, which correspond to the DNA sequences of the vectors pDONRTM221 (SEQ ID No: 11) and pENTTM221 (SEQ ID No: 12), were also used.
Competent E. coli cells were transformed with the recombinant expression vector pLenti6.4TM-CMV-V5-MSGW/miR (Life Technologies (Invitrogen)) and the cells were selected in ampicillin-supplemented LB medium. Colonies were picked and grown overnight in LB medium with ampicillin. Digestion with Hind was performed to verify a correct recombination. Lentiviruses (preferably pLenti6.4TM-CMV-MSGW/miR-XM-001056970-1919 (SEQ ID No: 21); pLenti6.4TM-CMV-MSGW/miR-XM-001056970-8223 (SEQ ID No: 24); pLenti6.4TM-CMV-MSGW/miR-XM-001056970-8531 (SEQ ID No: 20); pLenti6.4TM-CMV-MSGW/miR-neg (Life Technologies (Invitrogen)) were used to transfect HL-1 cardiomyocytes and NRVM.
The day before transfection, 293T human embryonic kidney cells were seeded. The lentiviral transfer vector (pLenti6.4-CMV-MSGW/miR, 6 μg) (SEQ ID No 9), the viral envelope plasmid (pMD-G-VSV-G, (Sigma-Aldrich), 2 μg), and the packaging construct (pCMV-ΔR8.2 (SEQ ID No: 22), 4 μg) were mixed with 150 mM sodium chloride and the mixture was added to a solution called “Polyplus transfection”. The mixture was incubated at room temperature for 20 min. This solution was added dropwise to the 293T cells which were incubated for 16 hours at 37° C. and with 5% CO2. The next day, the transfection solution was removed, and medium without FBS was added to the cells. After 48 hours of incubation, the supernatant was collected, and it was centrifuged and filtered through a 0.45 μm low-binding filter. The filtered supernatant was concentrated using Amicon filters according to the manufacturer's instructions. The various stocks that were generated, as well as the negative lentivirus control were titrated using the blasticidin method.
V. Generation of LRP1-Deficient Cardiomyocytes
In the transient transfection experiments, confluent NRVM were incubated for 18 hours in the presence of lentiviruses with 10 MOI. In stable transfection experiments, three days after infection with the virus, the HL-1 cells were treated with blasticidin (10 μg/mL). The medium was replaced with fresh medium containing blasticidin every 3 or 4 days until blasticidin-resistant colonies were identified. The clones with maximum inhibition of LRP1 expression were selected and grown.
13.2 Preparation of Human Smooth Muscle Cells from the Vascular Wall
The VSMC were obtained from human coronary arteries according to the explantation technique established by our group (Llorente-Cortés et al, ATVB 2000; Llorente-Cortés et al, Circulation 2002). To perform the experiments, VSMCs that had been thawed or subcultured between the 3rd and 7th passage. For treatment under normoxic conditions (21% O2), a Nirco incubator with an atmosphere of 74% N2 and 5% CO2 was used. For hypoxia, a Don Whitley Scientific Ltd. Anoxic Workstation H35de was used with a gas mixture of 1% O2, 94% N2, 5% CO2.
13.3 Isolation and Characterization of VLDL and LDL
The human VLDL (d1.001-d1.019 g/mL) and LDLs (d1.019-d1.063 g/mL) were obtained from a pool of sera from normolipidemic volunteers. The VLDL and LDL preparations used for the experiments were always less than 24 hours old and with no detectable levels of malonaldehyde or endotoxin. The modification of LDL by aggregation was performed mechanically (Llorente-Cortés et al, ATVB 1998). Native LDLs were brought to a concentration of 1 mg/mL by diluting them in PBS buffer, whereupon the preparation was vigorously stirred using a vortex mixer at room temperature (RT) for 4 minutes. Subsequently, the preparation was centrifuged at 10000×g at RT for 10 minutes to precipitate agLDL, and the supernatant was discarded. The agLDL were resuspended in PBS up to a protein concentration of 1 mg/mL. The agLDL obtained by centrifugation has a structure and functionality similar to that produced by incubation with versican proteoglycans of the extracellular matrix (Llorente-Cortés V et al, 2002 ATVB).
13.4 Measurement of the LRP1 Protein's Stability
In order to measure the stability of the protein LRP1, cycloheximide, an inhibitor of translation in eukaryotes, was used. VSMCs were pre-exposed to agLDL (100 μg/mL) for 18 hours before the addition of cycloheximide (100 μM). The cells were then collected at the following times after treatment with cycloheximide (6, 12, 24 and 32 hours), were harvested in lysis buffer, and then LRP1 protein concentration was analyzed using Western blot. The stability of the LRP1 protein was determined as the proportion of initial protein that remained at each time after treatment with cycloheximide.
13.5 Immunoprecipitation of the Ubiquitinated LRP1 Protein
The polyubiquitinated proteins were immunoprecipitated using the Ubiquitinated Protein Enrichment Kit (Calbiochem 662200). One aliquot of the protein extract was applied to poly-ubiquitin-enriched beads, and another aliquot was applied to negative control beads. After immunoprecipitation, the membranes were incubated with anti-LRP1 antibodies (Epitomics, 2703-1, dilution 1:7000) and with anti-ubiquitin antibodies (Calbiochem, 662099, dilution 1:5000).
13.6 Production of Polyclonal Antibodies
I. Synthesis of Peptides:
Three peptides were designated on the basis of sequence cluster II of alpha chain of LRP1 (SEQ ID No: 19). The sequences of these peptides and their locations are:

    
    
        Peptide 1 (P1): CTNQATRPPGGSHTDE (SEQ ID No: 13). S replaces the C of the original human LRP1 sequence (1051-1066).
        Peptide 2 (P2): DSSDEKSSEGVTHVC (SEQ ID No: 14). S replaces the C of the original human LRP1 sequence (1090-1104).
        Peptide 3 (P3): GDNDSEDNSDEENC (SEQ ID No: 15). S replaces the C of the original human LRP1 sequence (1127-1140).
    
    


These peptides were synthesized in the Separative Techniques and Peptide Synthesis Unit, at the University of Barcelona, through a solid-phase method using a peptide synthesizer. Peptides were then purified by high-performance liquid chromatography (HPLC) using UV detection at 254 nm. The purified peptides were characterized using mass spectrometry. The peptides were then coupled to the carrier KLH for immunization and to the carrier albumin to test the antibodies generated using the ELISA technique.
II. Immunization of Animals:
Production of polyclonal antibodies was performed at the Department of Cell Culture, Antibody Production and Cytometry, at the Universidad Autónoma de Barcelona (UAB). The animal study was approved by the Animal Research Committee of the UAB and by the Catalan government. For each peptide, two groups of three Balb/c mice (8 weeks old) were immunized intraperitoneally with 50 micrograms of the peptides conjugated to KLH. A small amount of blood was drawn to test for the presence of antibodies against the peptides using the ELISA method.
The mouse serum was collected following the immunization guidelines. The specific antibodies for each peptide were purified by immobilizing the peptides in SulfoLink affinity columns according to the manufacturer's instructions, and their levels were analyzed using ELISA.
III. ELISA
The levels of specific antibodies in the serum samples as well as those of the purified anti-peptide antibodies were determined using ELISA. Briefly, peptides conjugated to BSA and BSA alone were immobilized in 96-well plates at a concentration of 1 μg/mL in 0.1 M carbonate buffer and pH 9.6 for 60 minutes. After blocking with 1% BSA in PBS, the plates were incubated with primary antibodies at different dilutions, followed by incubation with peroxidase-linked secondary antibody. The enzymatic reaction was performed in a Sigma-Fast OPD device. After 30 minutes, the optical density was measured at 450 nm on a plate reader.
13.7 Lipid Extraction and Determination of Esterified Cholesterol (CE), Free Cholesterol (FC) and Triglyceride (TG) Content
Following incubation with VLDL, the cells were exhaustively washed and collected in NaOH. In the case of myocardial tissue, 5 mg of tissue were weighed, homogenized and triturated in NaOH. The cell or tissue homogenate was used for lipid extraction through the Bligh and Dyer method with minor modifications. Lipid extraction was performed by adding a mixture of methanol/dichloromethane (2:1, vol/vol). After evaporation of the organic solvent, the extract was dissolved in dichloromethane and used for thin-layer chromatography (TLC). TLC was performed on G-24 silica plates. A mixture of standards (cholesterol, cholesterol palmitate, triglycerides, diglycerides and monoglycerides) were applied on each plate. The chromatographic solution was heptane/diethylether/acetic acid (74:21:4, vol/vol/vol). The CE, FC and TG bands were quantified through densitometry against a standard curve of cholesterol palmitate, cholesterol, and triglycerides, respectively, using a densitometer.
13.8 Preparation of Doubly-Labeled VLDL
To prepare doubly-radiolabeled VLDL, a layer of cholesteryl-1,2[3H]-(N)(125 μCi) and trioleate glycerol [14C] was formed in a round-bottom bottle by evaporating the organic solvent at 37° C. under vacuum. Then, 50 mL of human plasma were added and the bottle was left rotating in a water bath at 37° C. for 30 hours. VLDL were isolated by sequential ultracentrifugation, dialyzed in PD10 columns, and filtered. The specific activity of the VLDLs was 492 cpm/μg of protein for [3H] and 102 cpm/μg of protein for [14C].
13.9 Determination of VLDL-[3H]-CE and VLDL-[14C]-TG Uptake by the Cardiomyocytes
LRP1-deficient or quiescent HL-1 control cells were incubated with doubly-radiolabeled VLDL (1.8 mM, 18 hours). At the end of the incubation period, the cells were washed and collected in 0.1 M NaOH. The radioactivity counts in 50 μL of cell homogenate were determined in a scintillation counter (Beckman Coulter, LS6500) and the counts (dpm) were normalized by the cellular protein.
13.10 Analysis of the Expression of mRNA and Protein
For the extraction of both mRNA and protein, the cells or tissue were homogenized in the TriPure isolation reagent following the manufacturer's instructions.
I. Expression of Messenger RNA (mRNA)
LRP1 mRNA, VLDLR mRNA, LDLR mRNA and CHFR mRNA expression was analyzed with real-time PCR using the ABIPRISM PCR-7000 Sequence Detection System (Applied Biosystems) with the following assays-on-demand: LRP1 (Rn01503901 or Mm00464601_m1), VLDLR (Rn01498166_m1 or Mm00443281_m1), LDLR (Rn00598440_m1 or Mm01151339_m1) and CHFR (Hs00943495_m1). ARBP (Rn00821065_g1) and GAPDH (4326317E) were used as endogenous controls. The real-time PCR was performed with 1 μL of the product of reverse transcription in 10 μL of the PCR Master Mix with primers at 300 nM and probe at 200 nM. The Ct (threshold cycle) values were used to calculate the expression that was normalized by the Ct of the endogenous control.
II. Protein Expression
The protein was isolated using the TriPure method and resuspended in SDS. The membranes were incubated with monoclonal antibodies against LRP1 (β chain, clone 8B8, RDI61067) or VLDLR (Santa Cruz Biotechnology Inc, D-17, sc-11823), or CHFR (Cell Signaling, 4297, dilution 1:1000 dilution). In order to check protein loading, the membranes were stained with Ponceau and the membranes were incubated with antibodies against β-tubulin (Abcam, ab6046) in the case of HL-1 or with antibodies against GAPDH (Santa Cruz Biotechnology, Inc., sc-20357) in the case of NRVM.
Monoclonal antibodies against LRP1 were used for detection of the cytoplasmic chain of LRP1 (beta chain, cytoplasmic) through the Western blot technique. Therefore, these antibodies were not able to inhibit the binding capacity of alpha chain of LRP1 (extracellular).
13.11 In Vivo Model of Myocardial Ischaemia
Neutral lipid profile and lipoprotein receptor expression were analyzed in the myocardium of a group of male pigs (Crossbred commercial pigs (Landrace-Largewhite) in which acute myocardial infarction was induced by occlusion of the left descending coronary artery (LAD) for 1.5 hours (n=6) and compared with a control group (sham, intervention without occlusion, n=3) as detailed in previously published work (Vilahur G et al, J Thromb Haemost 2009; Vilahur G et al, J Mol Cell Cardiol 2011). The coronary artery was occluded by angioplasty with a balloon via femoral. The site of occlusion was distal to the first diagonal branch. Ninety minutes of occlusion results in large transmural infarcts associated with a remodeling process. Samples from the periphery of the necrosis zone in the septal zone (ischaemic zone) and the left ventricle in the remote zone with regard to the infarction (non-ischaemic zone) were taken and used for molecular analysis.
13.12 Patients
A total of 55 hearts explanted from 26 patients with idiopathic dilated cardiomyopathy (DCM) and 29 patients with ischaemic cardiomyopathy (ICM) who underwent transplantation at the Hospital de la Santa Creu i Sant Pau in Barcelona or at the Hospital La Fe in Valencia were collected. Patients were classified according to the criteria of the New York Heart Association (NYHA) and received treatment according to the guidelines of the European Society of Cardiology. The biochemical and ultrasound data of these patients are shown in Table 3. Informed consent was obtained from all patients in accordance with our institutional guidelines. Four healthy hearts were obtained from donors who suffered a traffic accident and were used as controls.
13.13 Immunohistochemistry
The myocardial samples were cut into appropriate blocks and immersed in a fixative solution (4% paraformaldehyde), embedded in OCT, cut into slices of 5 μm in thickness and placed on slides of poly-L-Lysine. Anti-LRP1 antibody (Research Diagnostics, PRO61067) was used as a primary antibody. Prior to the incubation, the sections were washed and the endogenous peroxidase activity was inhibited with H2O2 using horse serum to inhibit non-specific activity. The primary antibody was detected with the avidin-biotin immunoperoxidase technique using a biotinylated secondary antibody (Vector). 3,3′-diaminobenzidine was used as a chromogen, and hematoxylin was used for nuclear staining. The protocol for VLDLR staining (Santa Cruz Biotechnology, sc-18824) was similar, except that permeabilization before applying the primary antibody was greater. The images were captured with a Nikon Eclipse 80i microscope and digitized with a Retiga 1300i Fast camera (240× magnification).
13.14 Cardiomyocyte Cell Cultures for the Production of Monoclonal Antibodies and Small Molecules for Use in In Vivo Models of Acute Myocardial Infarction
The HL-1 culture cell line was used as a model of adult cardiomyocytes. This cell line was kindly provided by Dr. W. C. Claycomb (Louisiana State University Medical Center, New Orleans, La., USA) and has phenotypic characteristics similar to those of adult cardiomyocytes (Claycomb W C et al, PNAS 1998). The HL-1 cells shall be kept in Claycomb medium (JRH Biosciences, Lenexa, Kans., USA) with foetal bovine serum (10%), norepinephrine (100 U/mL) and antibiotic. Before seeding, the wells shall be previously covered with gelatin and fibronectin, and the HL-1 cells shall be grown in parallel under normoxic and hypoxic conditions in the absence or presence of lipoproteins (Castellano J et al. JMCC 2011*). At this time, our group has stable LRP1-deficient cardiomyocyte lines generated by transfection with lentiviruses containing miRNA sequences that inhibit LRP1 expression (pLenti6.4-CMV-MSGW/miR, Invitrogen). We will compare the effect of the soluble minireceptors and of the antibodies generated, in order to prevent the transfer of esterified cholesterol from VLDL to the hypoxic cardiomyocytes.
13.15 Induction of Acute Myocardial Infarction to Study the Role of LRP1 Receptor Inhibition in Cardioprotection in In Vivo Models of Acute Myocardial Infarction
I. Mouse—
For the mouse model, apoE3leiden (E3L) transgenic mice are chosen, whose dyslipidemia pattern resembles that of patients with dysbetalipoproteinemia, in which the elevation of both cholesterol and triglycerides is limited to the VLDL. The mice were endotracheally intubated and connected to a fan following intraperitoneal anesthesia with a mixture of ketamine (50 mg/mL) and xylazine (2%). A left thoracotomy was performed between the 4th and 5th intercostal space to access the heart. The left coronary artery was occluded with a 5-0 suture thread, approximately 2 mm from the origin, between the edge of the left atrium and the pulmonary artery groove; the threads were crossed to completely stop the flow without damaging the artery. The occlusion was visually confirmed by the color change of the affected myocardium, and the disoclussion was verified by the disappearance of the epicardial cyanosis area.
II. Pig—
The pig model was ideal as a preclinical model in cardioprotection since the collateral coronary circulation and arterial anatomy of pigs and humans are quite similar. Furthermore, infarct size was fairly predictable. Induction of infarction in the pig model was performed as previously described in our center (Vilahur G et al, JMCC 2011). Twelve hours before the experimental induction of acute myocardial infarction (AMI), a loading dose of clopidogrel (150 mg/kg) was administered. The anesthetic was given by intramuscular injection of Zoletil® (7 mg/kg), Dormitorio® (7 mg/kg) and atropine (0.03 mg/Kg). The animals were subjected to endotracheal intubation and anesthesia was maintained by inhalation of isoflurane (1.5-2%). Continuous infusion of amiodarone (300 mg, 75 mg/h) starts at the beginning of the procedure in all pigs as a prophylaxis for malignant ventricular arrhythmias. Angiography images were used to guide the placement of balloon angioplasty (below the first diagonal branch). Balloon angioplasty (2.5-3 mm) was inflated to nominal pressure (complete occlusion of the coronary artery was verified by fluoroscopy). The occlusion was maintained for ninety minutes and the oxygen levels and electrocardiogram were monitored throughout the surgical procedure. The site of occlusion was located immediately distal to the origin of the first diagonal branch.
13.16 Release of Antibodies
Antibodies were intraperitoneally injected during surgery. In a pilot study, an analysis of the antibody dose required to inhibit VLDL-derived intracellular cholesteryl ester accumulation in ischaemic cardiomyocytes was performed.
13.17 Study of Infarct Size
I. Mouse

    
    
        To determine infarct size, the animals are killed and the heart mounted on a Langendorff apparatus to perfuse the heart with 10% formalin. After fixing, the scarring area of the sections stained with Masson trichrome (Sigma Aldrich) are measured with Bioquant imaging software and shall be expressed as a percentage of the total area of the left ventricle.
    
    


II. Pig—
After death, hearts are cut into sections of 5 mm in thickness from the apex to the atrioventricular groove. Alternative sections will be used for staining with triphenyl tetrazolium chloride (TTC) and Molecular Biology procedures. Blood from these animals is also collected from these animals prior to coronary artery occlusion and prior to killing to detect markers of myocardial necrosis such as troponin-I and CK-MB/CK.
13.18 Echocardiographic Parameters
Transthoracic echocardiography is performed before and after surgery using the Philips iE33 imaging system for pigs and Vevo 2100 (VisualSonics Inc.) equipped with a 30 MHz probe for mice.
13.19 Techniques Related to the Handling of Lipids and Lipoproteins
I. Lipoprotein Isolation.
VLDL and LDL are obtained by sequential ultracentrifugation at 4° C. in a dense KBr solution from plasma of normolipidemic donors. The lipoproteins are dialyzed and their purity is analyzed by agarose gel electrophoresis as previously described by the authors (Llorente-Cortés V et al, ATVB 2002*; ATVB 2006*).
II. Determination of Free Cholesterol, Cholesteryl Ester and Triglyceride Content in Cell and Tissue Samples.
Cell samples are collected in NaOH and tissue samples are crushed and exhaustively weighed before performing the lipid extraction and the thin layer chromatography of the samples as described previously by the authors (Llorente-Cortés V et al, ATVB 2002*, ATVB 2006*).
III. Preparation of [3H]CE-[14C]TG-VLDL and Determination of CE and TG Uptake by Cardiomyocytes
VLDL is doubly labeled using a method previously described, with minor modifications. The cholesteryl-1,2,-3H—(N) (125 μCi) and trioleate glycerol [14C] (25 μCi) (Perkin Elmer Life Sciences) are deposited in a flask with a round bottom to evaporate the organic solvent at 37° C. under vacuum. Then 50 mL of human plasma are added to the flask and allowed to rotate in a water bath under vacuum for 20 hours at 37° C. Thereafter, radiolabeled VLDL are isolated by ultracentrifugation, dialyzed, filtered and stored at 4° C. and protected from light until use.
The ability of the generated monoclonal antibodies to inhibit the uptake of labeled VLDL is analyzed in cell cultures. Uptake is determined by measuring the radioactivity of an aliquot from the cell suspension using an LS6500 scintillation counter. The cpm obtained are normalized by the cellular protein.
13.20 Molecular Biology Techniques
I. Obtention of Tissue Samples
Control mouse hearts are removed and treated. One sample from the ventricle is used for Molecular Biological studies and another for immunohistochemistry.
II. Gel Electrophoresis and “Immunoblotting” (for Analysis of Protein Expression)
Protein expression is studied by electrophoresis in SDS-polyacrylamide gels. The protein is transferred to nitrocellulose filters that have been incubated with antibodies specific to the genes under study (Llorente-Cortés V, Circulation 2002*).
III. Real-Time PCR
(for analysis of gene expression). Total RNA is isolated from the samples collected in TriPureTM reagent according to the manufacturer's instructions. The genes of interest are analyzed through real-time PCR after cDNA synthesis performed from 1 μg of RNA using the high capacity archive kit (Llorente-Cortés et al, J Mol Biol 2006*).
IV. Protein Immunoprecipitation Assays
This technique is used to study the formation of complexes in the intracellular signaling process. The cells are lysed in lysis buffer with protease inhibitors. Lysates are cleansed of debris by centrifugation at 10,000×g for 5 min at 4° C. The protein is then incubated with primary antibodies and then with agarose bound to G proteins overnight at 4° C. The immunoprecipitates are collected by centrifugation at 10,000×g for 5 min at 4° C., resuspended, and washed in lysis buffer. Subsequently, the immunoprecipitates are resuspended in Laemmli loading buffer and analyzed through Western blot.
V. Detection of the Phosphorylation of the Cytoplasmic Chain of LRP1
Anti-LRP1 antibody is used as an immunoprecipitation-inducing antibody, and anti-phosphotyrosine antibody is used as an antibody for the detection of the immunoprecipated complex in the transfer membrane (Boucher P et al, J Biol Chem 2002).
VI. Zymography
Metalloprotease activity is measured in the cell supernatants through a zymography using gelatin as a substrate. Equivalent amounts of supernatants are loaded onto 10% polyacrylamide gels containing gelatin at 1 mg/mL and are run at 4° C. The staining of the gels is performed with Coomassie R-250 blue and the gels are subsequently washed out with acetic acid (10%) and methanol (40%). The light bands on the blue background indicate the presence of proteolytic activity. These bands are quantified through densitometry (Otero-Viñas M et al, Atherosclerosis 2007*).
VII. Measurement of Oxidative Stress
The cell-permeable compound (H2DCF-DA) (non-fluorescent) is used to measure the intracellular levels of ROS. Within the cells, this compound is de-esterified at 2′,7′-dichlorofluorescein (H2DCF) which upon oxidation by ROS leads to the fluorescent compound (DCF) which remains inside the cells. The H2DCF-DA dissolves in DMSO and is added to the cells. The cells are incubated with the compound for 40 min at 37° C. Then the cells are collected and the fluorescence intensity is determined through flow cytometry.
LIST OF SEQUENCES
SEQ ID No: 1: top oligonucleotides designed to inhibit LRP1 expression (miR RNAi-XM—001056970—1919 or RNAi1919)
SEQ ID No: 2: bottom oligonucleotide designed to inhibit LRP1 expression (miR RNAi-XM—001056970—1919 or RNAi1919)
SEQ ID No: 3: top oligonucleotides designed to inhibit LRP1 expression (miR RNAi-XM—001056970—8223 or RNAi8223)
SEQ ID No: 4: bottom oligonucleotides designed to inhibit LRP1 expression (miR RNAi-XM—001056970—8223 or RNAi8223)
SEQ ID No: 5: top oligonucleotides designed to inhibit LRP1 expression (miR RNAi-XM—001056970—8531 or RNAi8531)
SEQ ID No: 6: bottom oligonucleotides designed to inhibit LRP1 expression (miR RNAi-XM—001056970—8531 or RNAi8531)
SEQ ID No: 7: top oligonucleotides designed for universal negative control (pcDNATM6.2-GW/miR-neg)
SEQ ID No: 8: bottom oligonucleotides designed for universal negative control (pcDNATM6.2-GW/miR-neg)
SEQ ID No: 9: vector pLenti6.4TM-CMV-MSGW SEQ ID No: 10: linear vector pcDNATM6.2_GW/miR
SEQ ID No: 11: vector pDONRTM221
SEQ ID No: 12: vector pENTRTM221
SEQ ID No: 13: peptide P1, amino acid sequence of cluster II of the alpha chain of the protein LRP1. Within this sequence, the zone of cluster II is identified: 1N7D, coded by the sequence located in the base interval: [1051-1066].
SEQ ID No: 14: peptide P2, amino acid sequence of cluster II of the alpha chain of the protein LRP1. Within this sequence, the zone of cluster II is identified: 1N7D Interface, coded by the sequence located in the base interval: [1090-1104].
SEQ ID No: 15: peptide P3, amino acid sequence of cluster II of the alpha chain of the protein LRP1. Within this sequence, the zone of cluster II is identified: 1N7D Interface, coded by the sequence located in the base interval: [1127-1140].
SEQ ID No: 16: RNAi1919, pcDNA6.2-GW/miR-XM—001056970-1919.
SEQ ID No: 17: RNAi8223, pcDNA6.2-GW/miR-XM—001056970-8223.
SEQ ID No: 18: RNAi8531, pcDNA6.2-GW/miR-XM—001056970-8531.
SEQ ID No: 19: Amino acid sequence of cluster II of the alpha chain of the protein LRP1. Within this sequence are identified P1 (coded by the sequence located in the base interval [1051-1066]), P2 (coded by the sequence located in the base interval [1090-1104]) and P3 (coded by the sequence located in the base interval [1127-1140]).
SEQ ID No: 20: pLenti6.4-CMV-MSGW/miR—XM—001056970—8531.
SEQ ID No: 21: pLenti6.4-CMV-MSGW/miR—XM—001056970—1919.
SEQ ID No: 22: packaging construct pCMV delta R8.2
SEQ ID No: 23: pLenti6.4/R4R2/V5-DESTTM
SEQ ID No: 24: pLenti6.4TM-CMV-MSGW/miR-XM-001056970-8223

## Claims

1. A peptide, wherein the peptide is selected from the group consisting of:
peptide P1 (SEQ ID NO: 13), peptide P2 (SEQ ID NO: 14), and peptide P3 (SEQ ID NO: 15).

2. A method for obtaining an antibody, comprising immunizing a subject with an antigen comprising the peptide of claim 1 and obtaining the antibody.