Endothelial Function: Cholesterol and Nitric Oxide Metabolism

Structure and Sources of Cholesterol

• Chemical structure of cholesterol:
  • Cholesterol is a sterol with amphipathic properties.
  • Contains a polar hydroxyl ($OH$) group.
  • Contains a non-polar steroid nucleus and a hydrocarbon side chain.
  • Widely distributed in all animal tissues throughout the body, but does not occur in plants.
• Sources of cholesterol in the body:
  • Endogenous source: Synthesized internally within the body.
  • Exogenous source: Obtained from dietary lipids.

Synthesis of Cholesterol

• Primary site of synthesis:
  • The liver is the major organ for cholesterol synthesis, accounting for $80\%$ of total endogenous synthesis.
• Precursor and general pathway:
  • Acetyl-CoA derived from the breakdown of glucose serves as the precursor.
  • The biosynthetic pathway consists of $11$ enzyme-catalyzed reactions.
• Rate-limiting reaction:
  • The $3\text{rd}$ reaction in the pathway is the rate-limiting step.
  • Catalyzed by the enzyme HMG-CoA reductase.
  • Converts $3\text{-hydroxy-3-methylglutaryl-CoA (HMG-CoA)}$ to mevalonate.
• Step-by-step enzymatic reactions:
  • Reaction 1:
    • $2\,\text{Acetyl-CoA} \xrightarrow{\text{Thiolase}} \text{Acetoacetyl-CoA} + \text{CoA}$
  • Reaction 2:
    • $\text{Acetoacetyl-CoA} + \text{Acetyl-CoA} + H_2O \xrightarrow{\text{HMG-CoA synthase}} \text{HMG-CoA} + \text{CoA}$
  • Reaction 3 (Rate-limiting step):
    • $\text{HMG-CoA} + 2\,\text{NADPH} + 2H^+ \xrightarrow{\text{HMG-CoA reductase}} \text{Mevalonate} + 2\,\text{NADP}^+ + \text{CoA}$
  • Reaction 4:
    • $\text{Mevalonate} + \text{ATP} \xrightarrow[\text{Mg}^{2+}]{\text{Mevalonate kinase}} \text{5-Phosphomevalonate} + \text{ADP}$
  • Reaction 5:
    • $\text{5-Phosphomevalonate} + \text{ATP} \xrightarrow[\text{Mg}^{2+}]{\text{Phosphomevalonate kinase}} \text{5-Pyrophosphomevalonate} + \text{ADP}$
  • Reaction 6:
    • $\text{5-Pyrophosphomevalonate} + \text{ATP} \xrightarrow{\text{Pyrophosphomevalonate decarboxylase}} \text{Isopentenyl pyrophosphate} + \text{ADP} + P_i + CO_2$
  • Reaction 7:
    • $\text{Isopentenyl pyrophosphate (IPP)} \xleftrightarrow{\text{IPP isomerase}} \text{3,3-Dimethylallyl pyrophosphate}$
  • Reaction 8:
    • $\text{3,3-Dimethylallyl pyrophosphate} + \text{Isopentenyl pyrophosphate} \xrightarrow{\text{Cis-Prenyl transferase}} \text{Geranyl pyrophosphate} + PP_i$
  • Reaction 9:
    • $\text{Geranyl pyrophosphate} + \text{Isopentenyl pyrophosphate} \xrightarrow{\text{Trans-Prenyl transferase / Cis-Prenyl transferase}} \text{Farnesyl pyrophosphate} + PP_i$
  • Branching pathways from farnesyl pyrophosphate:
    • Farnesyl pyrophosphate serves as a precursor for the synthesis of ubiquinone and dolichol.
  • Reaction 10:
    • $2\,\text{Farnesyl pyrophosphate} + \text{NADPH} + H^+ \xrightarrow[\text{Mg}^{2+}, \text{Mn}^{2+}]{\text{Squalene synthase}} \text{Squalene} + 2\,PP_i + \text{NADP}^+$

Excretion of Cholesterol

• Quantitative balance of cholesterol:
  • Normal dietary intake: Approximately $300\,mg/\text{day}$.
  • Endogenous body synthesis: Approximately $700\,mg/\text{day}$.
  • Total daily turnover/pool: Approximately $1000\,mg/\text{day}$.
• Excretion pathways:
  • Direct biliary excretion:
    • Approximately $500\,mg$ of cholesterol is excreted directly into bile as free cholesterol.
    • A portion of this biliary cholesterol is reabsorbed from the intestines via enterohepatic circulation.
    • Plant sterols present in vegetables inhibit intestinal reabsorption of cholesterol.
    • Unabsorbed cholesterol is metabolized by intestinal bacteria into fecal sterols, which are excreted in stool.
  • Bile acid conversion pathway:
    • Approximately $500\,mg$ of cholesterol is converted into bile acids.
    • Bile acids are excreted into bile as bile salts.
    • A fraction of bile salts is reabsorbed via enterohepatic circulation.

Regulation of Cholesterol Supply

• Mechanisms of cholesterol supply regulation:
  1. HMG-CoA reductase regulation:
     • Inhibition of HMG-CoA reductase directly reduces endogenous cholesterol synthesis.
  2. LDL receptor synthesis regulation:
     • Low intracellular cholesterol concentrations stimulate the synthesis of LDL receptors.
     • Increased LDL receptor expression enhances cellular uptake of cholesterol from the blood.
  3. Free cholesterol esterification:
     • Regulation of free intracellular cholesterol esterification with polyunsaturated fatty acids (PUFA) to form cholesterol esters.
     • Catalyzed by the enzyme Acyl-CoA: Cholesterol acyltransferase (ACAT).
     • Cholesterol esters are transported to the liver via high-density lipoprotein (HDL), reducing circulating blood cholesterol levels.

Normal Plasma Lipid Levels

• Total plasma lipids: $400\text{--}600\,mg/dL$
• Total cholesterol: $140\text{--}200\,mg/dL$
• HDL cholesterol:
  • Male: $30\text{--}60\,mg/dL$
  • Female: $35\text{--}75\,mg/dL$
• LDL cholesterol ($30\text{--}39\,\text{years}$): $80\text{--}130\,mg/dL$
• Triacylglycerols:
  • Male: $50\text{--}150\,mg/dL$
  • Female: $40\text{--}150\,mg/dL$

Classification and Causes of Hyperlipidemia

• Terminology:
  • Hypertriglyceridemia: Elevated concentrations of triacylglycerols in the blood.
  • Hypercholesterolemia: Elevated concentrations of cholesterol in the blood (associated with more severe clinical consequences).
  • Hyperlipidemia or Dyslipidemia: Generalized elevation of cholesterol, triacylglycerols, or both.
• Hypertriglyceridemia:
  • Predominant Hypertriglyceridemia:
    • An inherited condition commonly associated with excessive alcohol intake, Type 2 diabetes, and obesity.
    • Elevated triacylglycerol levels following meals are termed post-prandial hypertriglyceridemia.
    • Excessive intake of dietary fat in susceptible individuals can precipitate sharp increases in triacylglycerols, leading to acute pancreatitis.
  • Lipoprotein lipase deficiency:
    • Caused by a genetic defect in lipoprotein lipase.
    • Leads to hypertriglyceridemia accompanied by recurrent abdominal pain and acute pancreatitis.
    • Clinical manifestations include hepatosplenomegaly and eruptive xanthomas (yellowish subepidermal deposits of cholesterol and triacylglycerols).
• Mixed Hyperlipidemia:
  • An inherited disease characterized by hyperlipidemia combined with elevated LDL levels.
  • Frequently presents alongside Type 2 diabetes mellitus and obesity.
  • Causes simultaneous elevation of both cholesterol and triacylglycerols.
  • Serves as a major risk factor for cardiovascular disease.
• Hypercholesterolemia:
  • Familial Hypercholesterolemia:
    • A dominant genetic defect causing a deficiency in tissue LDL receptors.
    • Results in impaired cellular uptake of LDL and marked accumulation of LDL-cholesterol in the bloodstream.
    • Heterozygotes exhibit plasma LDL-cholesterol levels approximately $2\,\text{times}$ the normal average.
    • Homozygotes exhibit plasma LDL-cholesterol levels $3\,\text{to }5\,\text{times}$ the normal average.
  • Dietary High Cholesterol Ingestion:
    • Excessive cholesterol intake represses hepatic LDL receptor synthesis.
    • LDL receptor deficiency—whether genetic or dietary in origin—elevates blood cholesterol levels by decreasing cellular LDL uptake.
• Clinical conditions resulting in secondary hypercholesterolemia:
  • Diabetes mellitus: Increased fatty acid breakdown generates excess acetyl-CoA, which is directed into cholesterol synthesis.
  • Obstructive jaundice: Biliary clearance and excretion of cholesterol are blocked.
  • Hypothyroidism: Expression of hepatic HDL receptors is reduced, impairing effective cholesterol elimination.
  • Nephrotic syndrome: Urinary loss of albumin leads to a compensatory increase in globulins, including circulating lipoproteins.

Atherosclerosis Risk Stratification

• Total Cholesterol Risk Thresholds:
  • Low Risk: $<200\,mg/dL$
  • Borderline Risk: $200\text{--}240\,mg/dL$
  • High Risk: $>240\,mg/dL$
• LDL Cholesterol Risk Thresholds:
  • Low Risk: $<130\,mg/dL$
  • Borderline Risk: $130\text{--}160\,mg/dL$
  • High Risk: $>160\,mg/dL$
• HDL Cholesterol Risk Thresholds:
  • Low Risk: $>60\,mg/dL$
  • Borderline Risk: $35\text{--}60\,mg/dL$
  • High Risk: $<35\,mg/dL$
• Triglyceride Risk Thresholds:
  • Low Risk: $<200\,mg/dL$
  • Borderline Risk: $200\text{--}400\,mg/dL$
  • High Risk: $>400\,mg/dL$

Nitric Oxide Biochemistry and Functions

• General characteristics:
  • Nitric oxide acts as a paracrine hormone, producing biological effects directly within the target tissue where it is synthesized.
  • Three major biological functions:
    1. Blood vessel dilation
    2. Neurotransmission
    3. Immune response
• Isoforms of Nitric Oxide Synthase (NOS):
  • Endothelial Nitric Oxide (eNOS)
  • Neuronal Nitric Oxide (nNOS)
  • Inducible Nitric Oxide (iNOS)
• Biosynthesis and Mechanism of Action:
  • Enzymatic synthesis reaction:
    • Synthesized from the amino acid arginine by Nitric Oxide Synthase (NOS) to produce ornithine or citrulline and nitric oxide.
    • $\text{Arginine} + \text{NADPH} + O_2 \xrightarrow{\text{NOS}} \text{Citrulline} + NO + H_2O$
  • Cellular signal transduction:
    • Nitric oxide diffuses to target cells and binds to guanylate cyclase.
    • Activated guanylate cyclase catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP).
• Isoform-Specific Physiological Roles:
  • Endothelial Nitric Oxide (eNOS):
    • Produced within the endothelial lining of blood vessels.
    • Triggered by shear stress (e.g., exercise), acetylcholine, and bradykinin.
    • Promotes vascular smooth muscle relaxation, increasing blood flow.
    • Specific vascular functions:
      1. Vasodilation: Maintains normal blood pressure.
      2. Anti-inflammatory action: Prevents white blood cells (WBCs) from adhering to the vascular endothelium.
      3. Anti-thrombotic action: Prevents platelet aggregation and clot formation.
      4. Anti-oxidant action: Inhibits the oxidation of LDL.
  • Neuronal Nitric Oxide (nNOS):
    • Produced in central and peripheral nerves and the brain.
    • Triggered by intracellular calcium ($Ca^{2+}$) influx.
    • Functions as a neurotransmitter; mediates physiological responses such as penile erection.
  • Inducible Nitric Oxide (iNOS):
    • Produced in macrophages and neutrophils.
    • Triggered by inflammatory cytokines and bacterial endotoxins during infectious states.
    • Facilitates bacterial ingestion and intracellular killing by immune cells.
    • Pathophysiology: Systemic overproduction during severe bacterial infections leads to profound vasodilation, resulting in septic shock (a life-threatening medical emergency).

Stages of Endothelial Damage and Atherosclerosis

• Pathogenesis overview:
  • Deposition of cholesterol within the endothelium of capillaries and arterial walls leads to atherosclerosis, causing cardiovascular disease.
  • Predominantly affects the aorta, coronary arteries, and cerebral vessels.
• Stage I: Formation of Foam Cells:
  • Prolonged hypercholesterolemia leads to cholesterol accumulation beneath the arterial endothelial layer.
  • Oxidized LDL-cholesterol deposits within arterial walls and is engulfed by infiltrating macrophages.
  • Macrophages become engorged with cholesterol, transforming into foam cells—the pathognomonic hallmark of atherosclerotic plaques.
• Stage II: Progression of Atherosclerosis:
  • Lipid droplets accumulate within vascular smooth muscle cells inside the lesion.
  • Early-stage lesions remain reversible if LDL-cholesterol levels are reduced.
  • Continued accumulation of lipid droplets causes progressive, irreversible structural changes.
• Stage III: Fibrous Proliferation:
  • Activated macrophages and platelets release growth factors.
  • Lipoproteins, glycosaminoglycans, and collagen accumulate at the lesion site, driving a chronic inflammatory response.
  • Persistent arterial inflammation causes elevated plasma levels of high-sensitivity C-reactive protein (hs-CRP).
• Stage IV: Advanced Fibrous Plaque:
  • Proliferative cellular and matrix changes narrow the arterial lumen.
  • Lumen narrowing converts laminar blood flow into turbulent flow.
  • Turbulence increases the propensity for thrombus formation.
  • Structural arterial alterations become completely irreversible.

Myocardial Infarction and Ischemia Biochemistry

• Coronary artery occlusion and infarction:
  • Thrombus formation in a narrowed vessel can occlude major blood vessels.
  • Thrombosis within a coronary artery leads to myocardial ischemia and subsequent infarction (tissue cell death).
• Biochemical changes during ischemia in cardiac muscle cells:
  • Decreased blood supply forces cardiac tissue to shift to anaerobic glycolysis.
  • Anaerobic glycolysis increases dramatically, resulting in:
    • Decreased intracellular $ATP$ levels.
    • Increased intracellular $NADH$ levels.
    • Accumulation of lactic acid.
    • Decreased intracellular $pH$ (acidosis).
  • Physiological outcome: Inefficient heart muscle contraction, progressing to localized cardiac cell death if perfusion is not restored.
• Diagnostic modalities for cardiovascular disease:
  • Clinical history and physical examination.
  • Electrocardiogram (ECG).
  • Measurement of circulating cardiac biomarkers:
    • Troponin
    • Creatine kinase (CK)
    • C-reactive protein (CRP)