Biomarkers lecture

Cardiovascular and bone health

biomarkers
 • Biomarkers of Bone Health in CVD

•Both osteoporosis and cardiovascular disease (CVD) are major public health problems
leading to an increased morbidity and mortality rate. Growing data suggest the relationship
between osteoporosis and CVD, through mechanisms not fully elucidated but likely related
to common risk factors, common pathophysiological mechanisms, or both.

•In addition to menopause and advanced age, other risk factors for CVD, such as
dyslipidemia, hypertension, and diabetes, have also been associated with increased risk of
low bone mineral density (BMD).
• There are common underlying molecular pathways in the physiopathology of these two
diseases.

• Oxidative stress (OxS) and inflammation are key factors in both atherosclerosis and
osteoporosis.

• Elevated homocysteine (H-cy) concentration is associated with both CVD and

osteoporosis.

• Nitric oxide (NO), in addition to its known atheroprotective effect, appears also to play a
role both in osteoblast function and bone turnover.

• Impaired mineral metabolism appears to be associated with bone disease and also with
vascular calcification. Many traditional biochemical markers of bone remodeling retain
significance for atherosclerosis, CV risk, and vascular calcification.
• Gene deletions and variants have a pathogenic role in CVD and
vascular calcification as well as in osteoporosis.
•Prevention and treatment strategies for one of the two diseases may
be beneficial for the other one as both diseases share many risk
factors and comorbidities such as diabetes, chronic kidney disease
(CKD), hypertension, smoking, or low level of physical activity.
 • Cardiovascular Disease and Osteoporosis: Common Risk Factors and Common
Pathophysiological Mechanisms
• Indeed, there are interesting issues in bone loss and CVD in relation to gender, as estrogens
are protective for both conditions in premenopausal women.
• With regard to CVD, the lower risk for women has been reconsidered. In fact, after
menopause changes occur, and the risk of CV events exponentially rises, and the advantage
gap for women decreases, and becomes progressively smaller, until it overcomes the risk for
men in advanced age.
• Sex-related differences are also evident for bone, as bone mineral density (BMD) well
correlates with estrogen, and early postmenopausal bone loss is due to direct consequences of
estrogen fall.
• Moreover, bone loss is likely the main reason of BMD and risk of fracture in
female subjects after menopause and increases at a higher rate in elderly women
with respect to men.
• Conversely, men have higher bone density, and in older men BMD is more
dependent on BMD peak.
Main common risk factors for bone and cardiovascular disease:

Pathogenetic factors CV effects Bone effects

Aging – –
Male gender – (−/+) + (+/−)
Obesity – (−/+) + (+/−)
Hypertension – –
Diabetes – –
Dyslipidemia – −/+ (−)
Chronic kidney disease – –
Smoking – –
Physical activity + +
Inflammation – –
Oxidative stress – –
Nitric oxide + +
Homocysteine – –
OxLDL – –
Estradiol + +
• The relationship between obesity and the bone or the CV system is also
extremely complex, as adipose tissue is no longer considered an inert tissue
storing fat, but an active endocrine tissue that affects a variety of
pathophysiologic processes, including immunity and inflammation.
• Obesity is without doubt correlated with hyperinsulinemia due to insulin
resistance and to a low-grade chronic inflammatory state. Conversely, other
molecules, such as adiponectin, actively secreted by the adipose tissue, have
anti-inflammatory properties, and exert multiple protective effects on the (CV)
system. In particular, adiponectin also promotes bone remodeling through
positive effects on osteoblast proliferation and differentiation.
• Thus, the balance of the adipose tissue numerous biological effects likely
determines if consequences will be positive or negative. Given the association of
obesity with CVD, obese subjects are expected to develop adverse outcomes
after a cardiovascular event compared to individuals with normal body mass
index (BMI).
• However, many results evidenced a U-shaped relationship between obesity and
mortality from various diseases, including myocardial infarction and heart
failure (HF).
• These findings indicate that patients with higher BMI have better short and long-
term mortality rates, the so-called “obesity paradox” in CVD.
• On the other hand, a higher BMI exerts greater mechanical load on the bone,
which increases its BMD to compensate for this effect.
• Nonetheless, circulating levels of protective molecules, for instance, adiponectin,
are inversely correlated to adiposity in obese subjects.
• Many common pathogenic factors have been proposed to promote atherogenesis and
osteoporosis by acting on both vascular and bone cells. Pro-inflammatory cytokines such as
IL-6 and TNF-α have a recognized proatherogenic effect, and represent major factors for
activation of osteoclasts, showing stimulation of bone resorption and inhibition of bone
formation. Moreover, recent data from the Health Aging and Body Composition Study have
shown that elevated levels of these cytokines or their soluble receptors were related to an
increased risk of hip fractures in elderly subjects.
• The endothelial NO synthase signaling pathway, a key factor in the modulation
of atherosclerosis, has also a role in bone remodeling by decreasing osteoclast
formation through repression of the receptor activator of nuclear factor kappa-
B ligand (RANKL) as well as through reduction of the resorptive activity.
• Moreover, bone marrow (BM) is an important factor in the regulation of
myocardial remodeling, with eNOS of the BM identified as a regulator of
myocardial angiogenesis and fibrosis in pressure-induced cardiac hypertrophy.
FIND picture
• High level of homocysteine (HHcy) is a recognized risk factor for CVD, which also increases
osteoclast activity and decreases osteoblast activity with direct effects on bone matrix.

• The principal mechanisms by which HHcy may exert its actions included endothelial
dysfunction, reduced NO bioavailability, smooth muscle cell (SMC) proliferation,
mitochondrial abnormalities and OxS, and increased platelet aggregation.

• Hcy binds directly to the extracellular matrix (ECM) and reduces strength at the bone level.

• Recent experimental data on mice fed with a homocystine-supplemented diet, which leads to
severe HHcy, showed decreased bone quality and impaired fracture repair.
• Experimental data indicate that an atherogenic high-fat diet reduces bone
formation by blocking the differentiation of osteoblast progenitor cells.

• Moreover, very recent experimental data demonstrated that

hypercholesterolemia promotes the development of an osteoporotic bone
phenotype in mice, including an increase in osteoclasts, loss of trabeculae,
thinning of trabeculae and cortex, and reductions in failure load and energy to
failure.
• Both atherosclerosis and osteoporosis may result from estrogen deficiency after
menopause. It is well documented that estrogen deprivation after menopause
induced an increased high bone turnover and accelerated bone loss, with
upregulation of osteoclast formation and differentiation.
• On the other hand, postmenopausal hormone replacement therapy is beneficial on
bone metabolism and is considered a therapy for postmenopausal osteoporosis.
• Moreover, estrogen deficiency related to aging has been identified as one major
determinant of bone loss also in men.
• Bone and coronary arteries are target organs for estrogens, as shown by the
expression of estrogen receptors on osteoblasts, osteoclasts, and coronary artery
SMCs.
Thus, the intensity of estrogen deficiency would in turn activate different
estrogen targets involved in the regulation of postmenopausal osteoporosis,
atherosclerosis, and possibly arterial calcification as well, including inflammatory
parameters (cytokines and chemokines) and osteoclast regulators (OPG).

• In particular, reduced estrogens induce an increase in these molecules together

with a decrease in osteoclast regulators (OPG), a reduction in serum vitamin D
(vitD), increased inflammatory and oxidative processes, and reduced NO
bioavailability, key factors for the progression of bone loss and atherogenesis.
• Moreover, estrogens appear inversely related to levels of Hcy and lipids,
especially OxLDL. A loss in estrogen levels is associated with an increase in
parathyroid hormone (PTH), which accelerates the process of bone loss and
facilitates soft tissue calcium deposition, also in vessels.
• Mean platelet volume (MPV) as a reliable biomarker for
cardiovascular diseases. They reported that patients suffering from
acute myocardial infarction and stable coronary artery diseases
displayed higher MPV as compared to healthy controls. They
concluded that elevated MPV can be used as a biomarker for
monitoring risk of acute MI and CAD. Importantly, an increased
MPV also indicates a poor outcome among survivors of MI
suggesting that MPV may be a potential therapeutic target.
• Homocysteine, a homologue of cysteine, is synthesized from methionine by
removal of its terminal Cε methyl group. It was suggested to be a modest
independent predictor of coronary heart disease. Scientists found an
association between cardiovascular disease and elevated serum homocysteine
or the homozygous C677T mutation in methylene tetrahydrofolate reductase, a
gene that encodes an important enzyme in homocysteine metabolism.
Increased plasma homocysteine results from multiple factors, such as vitamin
deficiencies (B6, B9, and B12), renal impairment, and gene polymorphisms.
However, it remains debatable whether lowering homocysteine through
supplementing vitamin B6, B9, and B12 prevents cardiovascular disease.
• Serum magnesium is another biomarker for cardiovascular disease as they
detected elevated total cholesterol, triglycerides, very low-density lipoprotein
(VLDL) and LDL and reduced high DL cholesterol in subjects with lower serum
magnesium and CAD compared to controls. Diabetes, dyslipidaemia, and
hypertension were negatively correlated with serum magnesium levels.
Furthermore, dietary magnesium was positively correlated with serum
magnesium. The authors concluded that hypomagnesaemia and low dietary
magnesium in CAD patients are strong risk factors. Magnesium behaves like a
natural calcium channel blocker to attenuate blood pressure and blood pressure-
induced CAD. Supplementing dietary magnesium may improve hypertension
and CAD.
• Microalbuminuria (MA) is a persistent, increased urinary excretion of
albumin and based on the findings may also be a candidate biomarker for
cardiovascular disease risk. They found increased circulating levels of MA,
high-sensitivity C-reactive protein (hsCRP), and LDL cholesterol in MI
patients as compared to healthy individuals. They concluded that MA and hs
CRP evaluation may improve cardiovascular risk prediction when combined
with traditional lipid profiles. Similarly, other researchers reported that MA
also correlates with various cardiac abnormalities and diseases, such as left
ventricular dysfunction and hypertrophy, electrocardiographic abnormalities,
and ischemic heart disease.
Biomarkers of Bone Health in CVD
1-Cathepsin K

• Cathepsin K (CatK) belongs to the cysteine protease family originally identified as lysosomal enzymes.

• Specifically, CatK initially considered to be selectively expressed in bone, is the major protease of osteoclasts,
responsible for bone resorption, with proteolytic activities against several ECM components such as collagen I and II,
elastase, osteonectin, and OPN.

• More recent data have revealed additional roles for CatK in other pathological conditions, including cancer, and
autoimmune, infectious, and CVD.

• In fact, ECM remodeling is an important process in atherosclerotic plaque turnover and stability.

• CatK appears regulated by oscillatory shear stress, which may trigger a cascade of events leading to increased and
progressive atherosclerotic plaque.
Schematic representation of bone turnover biomarkers released during the activity of a basic
multicellular unit
 •CatK expression in normal arteries is low. However, CatK has been found in plaques
prone to rupture where it can mediate extensive matrix breakdown. Early human
atherosclerotic lesions showed CatK expression in the intima and medial SMCs, whereas in
advanced atherosclerotic plaques CatK appears mainly localized in macrophages and SMCs
of the fibrous cap.

• CatK has also been proposed to be a novel marker of adipogenesis and is involved in the
pathogenesis of obesity by promoting adipocyte differentiation.

• CatK is overexpressed in adipose tissues of obese subjects.

• Deficiency or selective inhibition of CatK reduces preadipocyte differentiation and

adiposity and increases glucose metabolism in mice under high-fat diet treatment.
 •In humans, significant elevation in the levels of CatK was observed in patients with
AMI (acute myocardial infarction).
•Moreover, CatK levels were found to be independent predictors of coronary artery
disease (CAD) and correlated positively with percent plaque volumes and inversely with
percent fibrous volumes by intravascular ultrasound.
•There is no doubt that CatK is a valid and attractive target for therapies against
osteoporosis but is also useful for other extra-skeletal diseases.
 2-Tartrate-Resistant Acid Phosphatase 5b (TRACP5b):

• Tartrate-resistant acid phosphatase 5b is a proteolytically processed isoform produced by

osteoclasts and represents a sensitive marker of bone resorption. This factor has been
found in osteoclast-like cells of monocyte-macrophage lineage in the atherosclerotic
plaque.

• It is independently associated with arterial stiffness together with the bone specific
alkaline phosphatase (BAP) and is a predictor of adverse CV outcomes in chronic kidney
disease patients.

• TRAP5b also represents an independent factor correlated with hs-CRP (high-sensitivity

C-reactive protein) and inflammation in hemodialyzed patients.
 3-Sclerostin:

• Sclerostin is a glycoprotein produced by osteocytes.

• This molecule inhibits the Wnt signaling pathway, regulating osteoblast activity and leading to decreased bone
formation. Higher serum sclerostin levels are associated with lower risk of fracture, higher BMD, and lower bone
turnover rate.

• Sclerostin is upregulated in experimental models of vascular calcification.

• Recent data from chronic kidney disease (CKD) patients showed higher sclerostin levels in patients with aortic
calcifications.

• Additional clinical and experimental studies are still needed to clarify whether sclerostin is an adverse or a
protective factor against progression of vascular calcification.
4- Fibroblast Growth Factor 23:

•Fibroblast growth factor 23 (FGF-23) is mainly produced by osteocytes and regulates phosphorus and vit
D metabolism.

•High FGF-23 levels promote left ventricular hypertrophy and are independently associated with greater
risk of CV events and mortality in CKD.

• High FGF-23 levels have been associated with vascular calcification and CV mortality, and with the
extent and severity of CAD in patients with CKD.

•Recent findings demonstrated that FGF-23 directly induces left ventricular hypertrophy, suggesting that
FGF-23 may not simply be a biomarker of CV risk but rather a key mediator of cardiac injury.
 5- Osteopontin: (OPN)

•OPN is produced by osteocytes, osteoblasts, and osteoclasts and represents a key component of the
mineralized extracellular matrices of bones.

•This protein retains both proinflammatory and anticalcific effects.

•It is upregulated in a variety of acute and chronic inflammatory conditions, including atherosclerosis,
where it is likely involved in the recruitment and retention of macrophages and T-cells toward the inflamed
sites.

•High levels of OPN mRNA and proteins were also reported in atherosclerotic plaques.

•OPN is independently associated with the presence and severity of CAD.

•OPN provides significant prognostic information in patients with chronic HF and it is upregulated after
AMI and stroke.
• OPN appears involved in left ventricle remodeling, and its production at sites of
tissue injury during the inflammatory and remodeling phases may modulate
neutrophil migration, macrophage recruitment and phagocytosis, regulation of
inflammatory gene expression, cardiac fibroblast adhesion and proliferation,
myofibroblast differentiation, ECM deposition, cardiac myocyte apoptosis and
hypertrophy, and angiogenesis.

• Conversely, increased circulating or valvular tissue levels of OPN in patients

with aortic valve calcification and stenosis have been found.
 6-Osteocalcin: (OC)
•A product of osteoblasts has attracted much attention as a hormone affecting glucose metabolism and fat
mass. In fact, based on experiments conducted on OC-deficient mice, it has been proposed that the
uncarboxylated form of OC is a pivotal factor by which bones influence insulin secretion and insulin action,
by increasing insulin release and sensitivity and energy expenditure, decreasing visceral fat.

•In humans, OC levels are associated to the degree of insulin resistance and to insulin release, and
also a beneficial effect of weight loss.

•Accordingly, in healthy elderly individuals, an inverse relationship was found between OC and
fasting plasma glucose, fasting insulin, and homeostasis model assessment-estimated insulin resistance
(HOMA-IR).
• In patients with type 2 diabetes (T2DM), serum OC levels were negatively
related to glucose and fat mass and atherosclerosis surrogates and positively
to total adiponectin levels.
• OC levels were inversely associated to the presence and severity of CAD, and
with fasting and post load (2 h) glucose and hemoglobin A(1c) values
• Low OC levels represented an independent risk factor for carotid
atherosclerosis in patients with T2DM, and inversely correlated with CRP.
 7- Osteoprotegerin (OPG):
•OPG is a glycoprotein, which binds the RANKL, and can reduce the production of osteoclasts
by inhibiting the differentiation of osteoclast precursors into mature osteoclasts.
•In epidemiologic studies, low OPG levels were related to higher prevalence of osteoporosis
and vertebral fractures.
•Based on experimental studies, OPG may be considered a protective factor for the vascular
system that prevents vascular calcification.
•Circulating levels of OPG correlate with CV risk factors, including hyperlipidemia, endothelial
dysfunction, diabetes mellitus, and hypertension.
•In particular, patients with clinical atherosclerosis had higher mean OPG, which remains
independently correlated with atherosclerosis also after multivariate adjustment for traditional
factors and CRP.
•OPG together with OPN levels is associated with arterial stiffness and the presence and
severity of CAD.
• Atherosclerotic risk factors, such as age, smoking, and new diabetes, were associated with increasing OPG
concentrations.

• In postmenopausal women without CVD, high OPG levels are positively related to markers of subclinical
arteriosclerosis (markers of endothelial function and arterial stiffness).

• In CAD, elevated OPG levels are associated to disease presence and severity. Accordingly, recent data suggest
that circulating OPG and OPN levels are positively associated with arterial stiffness, and the extent and
severity of CAD, independently of other known CV risk factors.

• OPG also correlates with the severity of peripheral artery disease, and appears associated with increased risk
of all-cause mortality, CVD mortality, and myocardial infarction in the general population and in ischemic
patients.
 8-Vitamin D:

•Vit D is a secosteroid hormone, recognized as a factor promoting bone growth and remodeling. In humans, the major
source of vit D is from exposure of skin to sun, with a smaller contribution from diet.

•Skin exposure to solar UV irradiation drives the conversion of 7-dehydrocholesterol to pre-vitamin D3, which is then
isomerized to vitamin D3 (cholecalciferol). Cholecalciferol, bounded to vitamin D-binding protein (DBP), enters into the
blood and first hydroxylates in the liver to 25(OH)D, and then to 1,25(OH)2D, the active hormone, by 1a-hydroxylase in the
kidney.
•Accordingly, a recent meta-analysis confirmed the inverse correlation of vit D with CV risk factors, including diabetes,
hypertension, and dyslipidemia. In clinical studies, low 25(OH)-vitamin D, 25(OH)D, was found to be associated with the
risk of ACSs, stroke, HF, and peripheral arterial disease, and prognostic for adverse event and in hospital mortality for ACS
patients.
•Also, obesity, a recognized risk factor for CVD, may be associated with a lower vit D status due to the lipophilic nature
of vit D and its sequestration in the fat.
 • Vit D also has a direct effect on calcium influx and affects myocyte contractility.
• Vit D has been also associated with vascular calcification, although some data support a protective rather
than an adverse effect. In addition, vit D affects NO levels, inflammatory parameters, angiogenesis, platelet
aggregation and insulin resistance and fasting glucose values.
• Recently, another mechanism by which vit D may exert its beneficial effects has been identified in the
antioxidant properties of this molecule, by inhibiting superoxide anion production and inducing NO release,
affecting mitochondria function, and cell viability, activating survival kinases.
• Other Factors

•A procollagen marker with higher prognostic potential is the N-terminal propeptide PIIINP, an index of
type III collagen turnover rate, higher levels of which predict an adverse outcome after a myocardial
infarction and in chronic HF.

•However, type I collagen is an important component of bone matrix, and osteoblasts produce its precursor,
the procollagen molecule, during bone formation. Procollagen type I N -propeptide (PINP) and procollagen
type I C-propeptide (PICP) are the extension peptides at each end of the procollagen molecule, indices of
type I collagen synthesis, and are cleaved by enzyme activity during bone matrix formation and released into
the circulation.

•The PICP has been found to predict adverse outcomes following myocardial infarction and in chronic HF.
Conversely, the N-terminal crosslinking telopeptides of type I collagen (NTx) and CTx are considered bone
resorption indices and represent proteolytic fragments of bone collagen matrix. ICTP is a fragment always
produced through action of matrix metalloproteinases at the C-terminal extremity of type I collagen, longer
than CTx.
• Peroxisome proliferators-activated receptors (PPARs)

• Peroxisome proliferators-activated receptors (PPARs) are a family of nuclear hormone

receptors, involved in the adjustment of cardiac lipid and glucose metabolism.

• Until now, three different PPAR subtypes have been identified, including PPARα, PPARβ and
PPARÿ. Some findings support the important regulatory role of the PPARα on β-oxidation,
whose expression level determines the utilization of fatty acids by cardiac tissues.

• Reduction of PPARα is a significant hallmark of cardiac hypertrophy, which contributes to

insufficient energy production and further progression of pathology.
• PPARs are critically involved in the regulation of a large number of genes that regulate
energy homeostasis, glucose triglyceride and lipoprotein metabolism, de novo lipogenesis,
fatty acid uptake, oxidation, storage and export, cell proliferation, inflammation and
vascular tissue function Because of their involvement in multiple metabolic processes.

• PPARs have been implicated in the pathogenesis obesity, Met S, diabetes, NAFLD and
atherosclerosis as such they represent important molecular targets for the development of
new drugs to treat these metabolic diseases.

• Because of their involvement in multiple metabolic processes, PPARs have been implicated
in the pathogenesis obesity, Met S, diabetes, NAFLD and atherosclerosis as such they
represent important molecular targets for the development of new drugs to treat these
metabolic diseases.
• H-FABP:H-FABP e A sensitive biomarker of early diagnosis and prediction of myocardial
damage:

• The heart-type FABP (H-FABP), also known as mammary-derived growth inhibitor, is probably the best-known
member of the FABP family.

• It is expressed in tissues with high demand of fatty-acids, such as heart, skeletal-muscle, brain, kidney, adrenal
gland, and mammary gland tissues, as well as in blastocysts.

• FABP3 was also found to be expressed in γ-aminobutyric acid (GABA)-ergic inhibitory interneurons of the male
anterior cingulate cortex in mice, suggesting that it has an important role also in cerebral PUFA-homeostasis.

• H-FABP itself is abundant in the cytoplasm of striated muscle cells and is rapidly released in response to cardiac
injury. H-FABP is expressed more abundantly in the heart’s ventricles and atria than in skeletal muscles or in
other organs.
• In healthy individuals, serum levels of H-FABP are in the single digit ng/ml range. Expression of H-
FABP is regulated by the microRNA miR-1, which might play a role in the progression of HF itself.

• Upon myocardial injury, H-FABP is rapidly released from myocytes into the systemic circulation,
due to its small size and free cytoplasmic localization.

• Also, transient increases in sarcolemmal membrane permeability are suspected to permit H-FABP
leakage into the systemic circulation. This so-called “wounding” of myocytes was observed, even
after short-term ventricular stress, and it may play an important role in diverse auto- and paracrine
mechanisms in the pathogenesis of HF.

• The elimination of H-FABP takes place via the kidney, explaining a shorter diagnostic window in
patients with normal renal function.

• It has been reported that H-FABP plasma levels returned to baseline within 20 hours after the onset
of symptoms in patients with acute myocardial infarction.
• Improved ability to rapidly differentiate severe such as acute myocardial infarction (AMI)
from pulmonary disorders in patients with chest pain and stuffiness onset will improve early
and targeted treatment of patients of the emergency department.

• H-FABP is a valuable marker of acute coronary syndrome, heart failure and pulmonary
embolism. During ischemia, H-FABP leaks out of myocardial tissue and its concentration in
blood increases within one hour and is reported to peak at about 3-6 hours and return to
normal baseline value in 20 hours.

• H-FABP plasma level is elevated faster than cardiac troponins, natriuretic peptides and
myoglobin. Thus to identify myocardial infarction and acute coronary syndrome, a
combination of H-FABP with troponin shows increased sensitivity over troponin alone in
patients with chest pain onset.
• A case report of a young man with three-vessel coronary artery disease (3vd) associated with familial
hypercholesterolemia (FH)

• A33-year-old man presented to the emergency department with the complaint of squeezing chest pain which
lasted for 6 hours. The patient underwent biochemical tests, an ECG, a coronary angiography, a chest X-ray, a
sonography of the heart and an examination of cardiologist.

• Results: In anamnesis the patient revealed that he had a stable angina pectoris for 2 months before this event.
Also, he had a positive family history for Familial Hypercholesteremia. First time hypercholesterolemia was
detected 5 months before this event and Rosuvastatin was prescribed. Blood tests showed elevated troponin
(65ng/l), total cholesterol (5,87mmol/l), LDL (3,91 mmol/l) levels, an ECG revealed myocardial ischemia. A
coronary angiography was performed and the diagnosis of 3 Vessel Disease was made. A coronary artery bypass
grafting surgery was urgently performed and symptoms of angina pectoris relieved. For further
hypercholesterolemia treatment Rosuvastatin and Ezetimibe were prescribed. After a few months a diagnosis of
Familial Hypercholesteremia was confirmed.