This document discusses HDL-C (high-density lipoprotein cholesterol), its structure, functions, and relationship to cardiovascular disease risk. It covers HDL-C composition, subfractions, origin, roles in cholesterol transport and inflammation regulation. The document challenges the traditional view of an inverse linear relationship between HDL-C levels and CVD risk, noting evidence for a U-shaped or J-shaped curve with higher risk seen at extremely high levels. It discusses assessing HDL function over static mass, including cholesterol efflux capacity, antioxidant activity, and effects on endothelial function markers. The document also reviews randomized trials of HDL-modifying drugs and conditions affecting HDL levels.

1. Dr. Prerna Goyal
Senior Consultant Physician
RG Stone and Super-specialty Hospital
Ludhiana
Understanding the Complex
HDL-C Biology and
Association with CVDs

2. Disclaimer
This presentation reflects the views
of the author only.

3. Content
HDL-C- Origin, Structure, composition and
Functions
HDL-C Structure – Function Relation
HDL-C and CVD Risk- transition from Inverse
Linear slope to U/J Curve
HDL-C numbers vs. Function –better CV
marker ?
Assessment of HDL-C Functions
Future therapeutic Implications

4. HDL-C Structure and
Composition
Highest relative density (1.063-1.21 g/ml)
Highest protein to lipid ratio
Size varies from 6.5 to 15 nm.
HDL particle- Proteins, lipids, microRNAs
(miRNA), and metabolites
A group of particles of varying size, densities, apo-
protein composition with marked structural,
physiochemical, compositional, functional and
biological heterogeneity

5. Application of –Omics to
Target HDLs
Discovery of genes, proteins, lipid species and
miRNAs
Use as biomarkers to improve identification,
treatment stratification and monitoring of
individuals at risk for CVDs and non CV diseases

6. HDL Proteome
Apo lipoprotein A-I –synthesized in liver and intestines,
70% of the total protein, present in almost all HDL-C
particles
Apo lipoprotein A-II - synthesized in liver, 20% of the total
protein, present in about two-third of HDL-C particles
Multiplicity of proteins- Apo III, IV, E, CI,II,III etc.
Functions- cholesterol hemostasis, lipid binding, immune
response, acute phase response (acute phase reactant
SAA4) , anti-oxidant and proteinase inhibition (α-1-
antitrypsin), hemostasis (α-2-HS-glycoprotein),
complement activation (eg, complement C3), and
inflammation (eg, haptoglobin-related protein)

7. HDL Lipidome
• PL and FC -the surface lipid monolayer, 30-40%
of total lipids
• CE and TG - the hydrophobic lipid core.
• Others- sphingolipids, steroids, triglycerides,
diacylglycerides, monoacylglycerides and FFAs

8. HDL Subfractions
Small, medium and large (S, M and L)-HDL
subclasses
OR
Two major subclasses-
 large buoyant (relatively lipid-rich) HDL2 particles
 Smaller, denser (relatively protein-rich) HDL3
particles
Further fractions into distinct subclasses upon
Non-denaturing polyacrylamide gradient gel
electrophoresis (GGE)- HDL3c, HDL3b, HDL3a,

10. HDL origin

12. HDL-C Functions
Pleiotropic properties
Cholesterol efflux capacity-
Antioxidant activity (acute phase reactant SAA)
Anti-inflammatory activity (haptoglobin-related
protein)
Antithrombotic activity, and
Prevents TNF-alpha induces apoptosis of
endothelial cell
Counteracts LDL-C oxidation

13. HDL-C Functions

14. Cholesterol Reverse
Transport
Role in the prevention of atherosclerosis, myocardial infarction,
transient ischemic attack and stroke.
Reverse transport mechanism to return cholesterol to the
liver from adipocytes, macrophages and endothelial cells.
ABCG1 and ABCA1 transporters- enable the transfer of
cholesterol to HDL.
LCAT- incorporates free cholesterol into the HDL-C particle
Uptake in liver- through three distinct pathways
 CETP pathway,
 LDL receptor pathway
 SR-B1 pathway

15. Cholesterol Reverse Transport- as a
biomarker for CVD Risk Prediction
Baseline CEC was significantly associated with incident
cardiovascular events independent of HDL-C and apoA-
I levels in the general population
ABCA1-dependent serum CEC correlated inversely with
pulse wave velocity, an index of arterial stiffness,
independent of HDL-C serum levels in healthy
individuals
E. Favari, N. Ronda et al, subjects,” Journal of Lipid Research, vol. 54, no. 1,
pp. 238–243, 2013.
S. Ebtehaj et al, Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 39,
no. 9, pp. 1874–1883, 2019.

16. HDL and Inflammation
HDLs and apoA-I have pro- and anti-inflammatory
effects.
The anti-inflammatory effects are beneficial in the
context of atherosclerosis and diabetes
The pro-inflammatory properties may contribute to
efficient clearance of bacteria in sepsis (also the reason
for limited success of HDL-raising therapies in reducing
ASCVD)
Dependent on cholesterol efflux.
Cholesterol efflux to HDLs and apoA-I is generally anti-
inflammatory in monocytes and macrophages.
However, excessive cholesterol depletion in

17. Anti-diabetic Properties of
HDLs
Improve glycemic control in animal models of diabetes
by enhancing pancreatic β-cell function and survival
and improving insulin sensitivity.
HDLs also inhibit β-cell apoptosis and protect β cells
from oxidation by LDLs.
Post hoc meta-analysis of CETP inhibitor trials- a 12%
reduction in incident diabetes.
Dalcetrapib trial- the reduced incidence in diabetes
may be a consequence of the treatment-related
increase in HDL-C and regression from diabetes to no
diabetes.
Masson W, Diabetes Metab. 2018; 44:508–513.
Schwartz GG, Diabetes Care. 2020; 43:1077–1084.

18. HDL-C Structure- Function Relation
Molecules Function in HDL-C Disecase Association
HDL-Particle number Determines CEC Inversely with risk of
ASCVD
Small HDL Determines CEC Inversely with risk of
ASCVD, positively with
diabetes
Large HDL Determines CEC Positively with Diabetes
Pre-B HDL Determines CEC Positively with ASCVD
Apo AI Activator of LCAT, Ligand
of HDL receptor and
ABCA1, antioxidative
Inversely with risk of
ASCVD
Apo CIII Promotes apotosis of
endothelial cells, inhibits
CEC
Positively with risk of
ASCVD and DM
Apo E Hepatic removal of HDL,
Promotes CEC, anti-
Inversely with risk of
ASCVD

19. HDL-C Structure- Function Relation
Molecules Function in HDL-C Disecase Association
Paraxonase 1 Inhibition of lipid
peroxidation
Inversely with ASCVD or
diabetes
Cholesteryl esters Core lipid determining size
and shape
Inversely with ASCVD,
diabetes and other diseases
Triglycerides Core lipid Positively with ASCVD
Sphingomyelins Determine rigidity of HDL
and thereby cholestrol effux
capacity and antiapoptotic
activity toward endothelial
cell
Inversely with diabetes or
ASCVD
Sphingosine-1 multiple vasoprotective
antidiabetic and anti-
inflammatory actions
Inversely with ASCVD or
diabetes
miE-223 Most abundant miRNA in
HDL; regulates VCAM
expression in endothelial
cells and cholesterol
Increased in ACS and
diabetes

20. HDL-C and CVD Risk- transition
from Inverse Linear slope to U/J
Curve
The HDL Hypothesis- “GOOD CHOLESTEROL” is no more
GOOD in all circumstances
First report in 1950s
1980s- The landmark epidemiological study, Framingham
Heart Study
There was a clear inverse relationship between HDL-C
concentrations and risk CHD, even at LDL-C below 70
mg/dL
Each increment of 1 mg/dL in HDL-C, the CHD risk is
reduced by 3% in women and by 2% in men

21. Challenges to Traditional Inverse
Linear Relation between HDL-C
and CVD
Observational cohort, Genome Wide association
studies - existence of a plateau effect or
elevated CVD risk and higher total mortality in
individuals with extremely high HDL-C levels
U-shaped association between HDL-C and CV
mortality /all cause mortality
HDL-C may be a double edge sword for
atherosclerosis.

22. HDL-C Numbers and CVD
Risk
CANHEART database (Canada)-
631 762 participants (55% women) age 57 years, follow-up 4.9 years.
Lower HDL-C was associated with higher risk of cardiovascular,
cancer and other mortality, and Higher HDL-C was associated with
higher risk of non-cardiovascular mortality.
Copenhagen General Population Study and the Copenhagen City
Heart Study-
52 268 men and 64 240 women (age 57 years, follow-up 6.0 years). U
shaped association with very high HDL cholesterol was most
pronounced in men and for cardiovascular mortality.
Madsen, C.M.; Two prospective cohort studies. Eur. Heart J. 2017
Ko, D.T.; The CANHEART Study. J. Am. Coll. Cardiol. 2016, 68, 2073–
2083.

23. HDL-C Numbers and CVD
Risk
Danish Study- increased risk of all-cause mortality
associated with extremely high HDL-C levels.
A multi-cohort study- CVD risk did not reduce further
with HDL-C values higher than 90 mg/dl in men and
75 mg/dl in women
A. Hirata et al, pooled analysis of 9 cohort studies : the EPOCH-JAPAN study,” Journal of
Clinical Lipidology, vol. 12, no. 3, pp. 674–684.e5, 2018.
J. T. Wilkins et al Journal of the American Heart Association, vol. 3, no. 2, article e000519, 2014.

24. Age, Ethnicity and Gender Specific
Relation between HDL-C and Mortality
Risk
Yi et al, International Journal of Epidemiology-
15 860 253 Korean adults (48% women, age 47 years, follow-up 8.6
years)
A clear U-shaped association for both men and women with a
tendency for the associations to be stronger for men than women.
The U-shaped relationship between HDL-C and all-cause mortality
was present for all age groups between 18 and 64 years However,
particularly at high HDL-C concentrations, the associations
attenuated for older individuals between 65 and 99 years of age.
Linear inverse association preserved <40 mg/dL in men and <50 to
58 mg/dL in women, no association across the normal range (40 to
96 mg/dL in men and 50 to 134 mg/dL in women), and a modest but
increased ASCVD risk at HDL-C levels >90 mg/dL in Asian
The link among the Black population may be attenuated or even
trend in the opposite direction compared with the White population.

25. Conditions Ass. With Altered
Levels of HDL-C

26. RCTs with Drugs Acting on HDL- C
Levels

27. HDL-C numbers vs. Function –
which is a better CV marker
Emerging consensus - HDL structural components
and functional aspects may be better predictors of
CVD risk than static mass of HDL measured
through HDL-C.
It is the QUALITY rather than quantity of HDL is
more relevant for its atheroprotective activity

28. Assessment of HDL
Functions
Cholesterol Efflux Capacity- using radioisotope
labelled cholesterol, fluorescently labelled cholesterol or
cell free assays (antibody/liposomes)
Antioxidative activity- measuring degree of LDL
oxidation via cell free assays (CFA) or LDL medicated
monocyte chemotactic activity (MCA) eg higher MCA
and CFA means proinflammatory and pro-oxidant even
with normal or higher HDL values
Endothelial eNOS and VCAM-1/ICAM-1 Assay-
measuring the production of nitric oxide by electron spin
resonance spectroscopy or by fluorescence-based
techniques in cell system or NO mediated vasodilatation

29. Assessment of HDL
Functions
Anti-apototic Activity- expression of caspase-
3 (as marker of apoptosis) by western blot or by
real-time PCR.
Future Goal:
• An assay for composite measure of HDL
function
• adaptable for clinical setup

30. Future Changes in HDL-C & CV Risk
Prediction Models
Existing CV risk evaluation tools-Framingham Risk Score
and American College of Cardiology/American Heart
Association (ACC/AHA), pooled cohort ASCVD risk
calculator -may incorporate or modify the HDL-C guidelines
for primary prevention of CVDs.
Recent European guidelines recommend not using HDL-C
as a risk measure in cases when HDL-C values exceed 90
mg/L
The HDL-C/LDL-C ratio may prove misleading in cases of
high HDL-C and comorbidities, including CHD, diabetes
mellitus and chronic kidney disease

31. Future Therapeutic
Directions

32. Future Therapeutic
Directions
Recombinant ApoA-I Milano infusions- caused a
significant regression of coronary atherosclerosis in
patients with ACS. In a pilot trial, MDCO-216 did not
produce a significant beneficial effect on CAD progression
measured by Intravascular Ultrasound (IVUS)
CSL112 (a reconstituted, infusible, plasma-derived
recombinant apoA-I and phosphatidylcholine) -In
phase 2 clinical trial, 4 weekly infusions of CSL112 among
patients with acute myocardial infarction reduced major
adverse cardiovascular events without any significant
alterations in liver or kidney functions

33. Future Therapeutic
Directions
Autologous delipidated HDL plasma infusions
Gene Therapy- recombinant adeno-associated virus
(AAV) technology
RVX-208 (apabetalone)- epigenetic modification
altering apoA-1 transciption, modest elevation in HDL-
C, doubtful clinical benefits in phase III trials
Recombinant LCAT: esterifies cholesterol in HDL-C.
Phase II trials, IV infusion, raises HDL-C by 50%

34. Final Inference
HDLs are diverse and carry a larger number of
proteins, lipids and miRNAs with diverse functions.
HDL-C as a CV risk marker should consider the
potential for nonlinearity as well as effects of ethnicity,
age, gender and HDL functionality.
HDL functionality plays a much more important role in
athero-protection than circulating HDL-C levels.
HDL functionality cannot be inferred from the plain
measurement of plasma HDL-C levels
HDL -CEC from macrophages is a key metric of HDL
functionality.

35. Final Inference
Future therapeutics will focus on new agents that
would be able to enhance CEC, improve HDL
functionality by altering its composition and
potentially decrease cardiovascular risk along with
a favorable side-effect profile
Understanding the complex nature of HDL and its
role as a protective agent, biomarker, and
therapeutic target in CVD remains an exciting area
of research.

37. Thanks

38. Anti-inflammatory to Pro-
inflammatory Shift
In ACS, protein content in HDL-C undergo
modifications
Increase in SAA and C3 atherosclerotic plaque
Alwaili, K.The HDL proteome in acute coronary
syndromes shifts to an inflammatory profile.
Biochim. Biophys. Acta Mol. Cell Biol. Lipids
2012,

39. Effects of Niacin on CVD Risk
Reduction
The Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High
Triglycerides: Impact on Global Health Outcomes (AIM-HIGH) trial-
3414 high-risk patients with CVD on statin therapy and low HDL-C levels (<40 mg/dl in
men and <50 mg/dl in women)
The trial was stopped early due to the lack of any additional clinical benefit of niacin over
statin in reducing the incidence of CVD events, despite significant improvement in HDL
cholesterol levels (HDL cholesterol level increased by 25.0% vs. 9.8% at 2 years in the
niacin versus placebo group, , respectively)
The Heart Protection Study 2-Treatment of HDL to Reduce the Incidence of Vascular
Events (HPS2-THRIVE) trial -in 25,673 adults with known atherosclerotic vascular disease
(on statins)
The addition of niacin-laropiprant had no significant reduction in major vascular events as
compared with placebo (13.2% and 13.7% of participants had a cardiovascular

40. Cholesteryl ester transfer protein (CETP)
inhibitors on CVD Risk Reduction
The first three trials with CETP inhibitors failed to
show any reduction in risk of CVD events, as a
result of which they were stopped.
- ILLUMINATE (torcetrapib)
- REVEAL (anacetrapib)

41. Both CETP inhibitors and niacin preferentially
increase the levels of the large HDL particles,
whereas their effects on the total number of HDL
particles are weaker

42. Genome Wide Association
Studies
o Three functional variants of hepatic lipase associated
with a modest rise in levels of HDL-C did not improve
cardiovascular risk
o Functional mutations in ATP-binding cassette
transporter A1 (ABCA1) leading to a 29.3% reduction
in HDL-C levels, did not adversely affect cardiovascular
risk
o Carriers of a single nucleotide polymorphism in the
endothelial lipase gene leading to an increase of HDL-C
levels by 5.4 mg/dL, did not enjoy a reduced risk of
myocardial infarction, as compared with noncarriers

43. The ApoA-I Milano Mutation-
Low HDL-c but Functional
First described in 1980 in a family originating from Limone
sul Garda, a small town outside Milan in northern Italy.
Single AA substitution of arginine to cysteine at the
position 173 in the primary sequence of ApoA-I.
Formation of homodimers (AI-M/AI-M) and heterodimers
with apolipoprotein AII (AI-M/AII).
Lipid Profile of carriers of the ApoA-I Milano mutation -very
low HDL-C levels and moderate hypertriglyceridemia but
without evidence of premature CAD or preclinical coronary
or carotid atherosclerosis

44. Structural Changes – Loss of
Functionality (Dysfunctional HDL-
C)
An increase in serum amyloid A1, serum amyloid A2, and
alpha-1 antitrypsin on HDL- attenuation of
atheroprotective functions by limiting its ability to promote
RCT and to prevent LDL modification
A decrease in apoA-I and paraoxonase 1- attenuation of
atheroprotective functions
Modification at tyrosine residue of apoA-I- impaired
ABCA1-dependent cholesterol transport
ApoA-I containing a 2-OH-Trp72 group (oxTrp72-apoA1)-
accounts for 20% of the apoA1 in atherosclerosis-laden
arteries.

45. Structural Changes – Loss of
Functionality (Dysfunctional HDL-
C)
A higher sphingomyelin to
phosphatidylcholine ratio- diminished HDL
antioxidative activity by altering the rigidity of
the surface monolayer
Protein carbamylation- contributes to foam
cell formation in atherosclerotic lesions
MPO-mediated oxidation of ApoA-I -has
also been found to impair HDL function in
regard to its RCT, antioxidant and anti-
inflammatory activities

46. HDL Structure-Function
Relationship
Apo lipoprotein M (apo M):
physiological carrier protein of S1P in HDL
partly responsible for anti-atherogenic effects,
ability to enhance cholesterol efflux from macrophage foam cells,
Anti-oxidative properties
Apo-AI –
Reflects the degree of cardiovascular protection.
An increase by 1 SD in the level of Apo-AI in apo-lipoprotein B-depleted
plasma decreased almost by half the risk of having ACS.
Mediates cholesterol efflux
Preventing LDL oxidation by contributing to inactivation and subsequent
transfer of lipo-peroxides

47. HDL-Particle Size and CVD
Risk
The IDEAL (Incremental Decrease in End Points through
Aggressive Lipid Lowering) trial
EPIC (European Prospective Investigation into Cancer
and Nutrition)-Norfolk case-control study-
very high plasma HDL-C levels (≥70 mg/dL) and very large
HDL particles (>9.53 nm) were associated with higher risk for
CVD.
Van der Steeg et al, The IDEAL and EPIC-Norfolk studies. J. Am. Coll. Cardiol.
2008,

48. HDL-Particle Size and CVD Risk
MESA cohort- small- and medium-sized HDL particles
were strongly and inversely associated with carotid intima
thickening
A large multiethnic study of patients without baseline
CHD- the concentration of HDL-C was no longer
associated with CIMT or CHD, whereas HDL-P remained
independently associated with both CIMT and CHD.
R. H. Mackey, Journal of the American College of Cardiology, vol. 60, no. 6, pp. 508–516,
2012.
S. Mora, J. D. Circulation, vol. 119, no. 7, pp. 931–939, 2009.
R. de Miranda Teixeira, Cardiology Research and Practice, vol. 2019, Article ID 3074602, 7

49. HDL Structure-Function
Relationship
HDL-bound sphingosine 1-phosphate (S1P)
Endothelial protection via the activation of nitric oxide
synthase in endothelial cells
Potent chemoattractant for endothelial cells and limit
abnormal vascular permeability via the stimulation of the
assembly of vascular endothelial (VE)-cadherin–
containing adherens junctions among endothelial cells
1-SD increment in S1P levels in apolipoprotein B-depleted
plasma was found to decrease ACS risk by 30%

50. Factors Altering HDL
Functionality
Acute-phase response state- creates an oxidative environment
which can convert HDL from an anti-inflammatory to a
proinflammatory particle
Glycation- may also impair HDL function, contributing factor to
the accelerated atherosclerosis in Type II diabetes mellitus
Metabolic syndrome- the small-dense HDL particles become
dysfunctional
Ethnicity- black South African women, in comparison to white
women, display improved HDL antioxidant functionality and are
relatively protected against CHD despite greater prevalence of
obesity and lower circulating HDL-C levels than white women.
Obesity/ Bariatric surgery
Dietary habits

51. The Origin of HDL
The formation of HDL-C starts in the liver and intestine.
First step- synthesis of main structural apolipoproteins, Apo-AI.
The secretion of lipid-poor protein is followed by the interaction of ApoA-I with the
cholesterol–phospholipid transporter ABCA1 (ATP Binding Cassette A1) in order to
acquire cholesterol and phospholipids, which results in the formation of nascent HDL-C
particle (pre-beta HDL).
In subsequent steps, HDL-C travelling in the circulation gains additional free cholesterol
and phospholipids from peripheral tissues, chylomicrons and very-low-density lipoprotein
(VLDL) and apolipoproteins coming from the hydrolysis of triglyceride-rich lipoproteins.
The core of mature HDL-C particles comprises cholesteryl esters (CE), which are formed
by LCAT acting on cholesterol at the surface of HDL-C and subsequently incorporated
[15]. Cholesteryl esters in HDL-C can be cleared either via direct uptake by the liver or
steroidogenic tissues in a process mediated by HDL-C receptor scavenger receptor B1
(SR-BI) or via plasma cholesteryl ester transfer protein (CETP)–mediated transfer to
apoB-containing lipoproteins, typically in exchange for triglycerides. It has been found that
HDL-C metabolism (which translates into plasma HDL-C concentration) is mutually
regulated by various enzymes, apolipoproteins, cell surface receptors and cellular lipid
transporters. The differences in HDL-C particles density, size and composition are
associated with the complexity of their metabolism. Observed plasma levels of HDL-C
mirror the net state of production, modifications and catabolism [2]. Apart from the
aforementioned processes, some genetic and environmental factors can also impact
HDL-C levels. For example, the presence of obesity, type 2 diabetes and inflammatory
state, as well as smoking, have been demonstrated to decrease HDL-C concentrations,
while exercise, oestrogen and thyroid hormone tend to raise its levels [

52. HDL-C Numbers and CVD
Risk
Both Low and high HDL-C concentrations associate
with a higher risk of cardiovascular as well as non-
cardiovascular mortality
HDL-C- more of a marker of general health than a
modifiable risk factor for cardiovascular disease
RRT hypothesis can account for high CV mortality but
reasons for non CV mortality ?