Role of vitamin C in the function of the vascular endothelium

Abstract

Significance: Vitamin C, or ascorbic acid, has long been known to participate in several important functions in the vascular bed in support of endothelial cells. These functions include increasing the synthesis and deposition of type IV collagen in the basement membrane, stimulating endothelial proliferation, inhibiting apoptosis, scavenging radical species, and sparing endothelial cell-derived nitric oxide to help modulate blood flow. Although ascorbate may not be able to reverse inflammatory vascular diseases such as atherosclerosis, it may well play a role in preventing the endothelial dysfunction that is the earliest sign of many such diseases.

Recent advances: Beyond simply preventing scurvy, evidence is mounting that ascorbate is required for optimal function of many dioxygenase enzymes in addition to those involved in collagen synthesis. Several of these enzymes regulate the transcription of proteins involved in endothelial function, proliferation, and survival, including hypoxia-inducible factor-1α and histone and DNA demethylases. More recently, ascorbate has been found to acutely tighten the endothelial permeability barrier and, thus, may modulate access of ascorbate and other molecules into tissues and organs.

Critical issues: The issue of the optimal cellular content of ascorbate remains unresolved, but it appears that low millimolar ascorbate concentrations are normal in most animal tissues, in human leukocytes, and probably in the endothelium. Although there may be little benefit of increasing near maximal cellular ascorbate concentrations in normal people, many diseases and conditions have either systemic or localized cellular ascorbate deficiency as a cause for endothelial dysfunction, including early atherosclerosis, sepsis, smoking, and diabetes.

Future directions: A key focus for future studies of ascorbate and the vascular endothelium will likely be to determine the mechanisms and clinical relevance of ascorbate effects on endothelial function, permeability, and survival in diseases that cause endothelial dysfunction.

FIG. 1.

Ascorbic acid metabolism. Ascorbate donates a single electron to become the ascorbate radical, which reacts with another ascorbate radical to form a molecule each of ascorbate and dehydroascorbate (DHA). The latter is unstable at physiologic pH and if not reduced back to ascorbate via GSH-dependent mechanisms, it will undergo irreversible ring opening and loss. In buffers, DHA forms a hemiketal that has a molecular structure resembling that of glucose.

FIG. 2.

Uptake and distribution of ascorbate across the vascular bed. Ascorbate (AA) is taken up from the intestine either on the SVCT1 or as DHA on glucose transporters (not shown). Once inside the intestinal epithelium, it exits by an unknown mechanism on the basolateral membrane into the interstitium and then into nearby capillaries. Ascorbate in the bloodstream is taken up by erythrocytes (either as DHA or as slow diffusion) and by leukocytes and endothelial cells on the SVCT2. Plasma ascorbate is distributed by the vascular tree to organ beds. Interstitial ascorbate is then taken up by the SVCT2 on nucleated cells in the organs. In the central nervous system, ascorbate enters the cerebrospinal fluid largely by secretion from the choroid plexus (not shown). SVCT1, sodium-dependent vitamin C transporter 1; SVCT2, sodium-dependent vitamin C transporter 2.

FIG. 3.

Endothelial cell ascorbate uptake and recycling. Ascorbate (AA) enters endothelial cells largely on the SVCT2, although a small amount may come in as DHA on glucose transporters (GLUT), to be rapidly reduced to ascorbate in the cell. Once in the cell, ascorbate can donate an electron ferric iron, superoxide (O₂^•−), and other radical species generated in mitochondria or via activation of cell surface receptors, such as those for thrombin or advanced glycation end-products (AGE). The resulting ascorbate radical (AA^•−) is mostly reduced directly back to ascorbate by NADH- and NADPH-dependent reductases. However, if the oxidative stress is overwhelming, the ascorbate radical may dismutate to form ascorbate and DHA, with subsequent reduction of the latter back to ascorbate.

FIG. 4.

Routes of transfer of ascorbate out of the vascular bed as represented by endothelial cells in culture on semi-porous filters. Ascorbate (AA) or DHA added on the luminal side of endothelial cells cultured on semi-porous membranes enter the cells on the SVCT2 or GLUT-type transporter, respectively. The resulting ascorbate is trapped with little exit on the basolateral side of the cells over a 90 min time-frame. Rather, most ascorbate passes between the cells by a paracellular route, which intracellular ascorbate tightens. There may also be some transit of ascorbate between the cells as sieving across tight junctional proteins.

FIG. 5.

Ascorbate recycling of BH₄ and preservation of nitric oxide. Dimeric eNOS (eNOS_d) attached to the endothelial cell plasma membrane utilizes arginine, molecular oxygen, and BH₄ to generate nitric oxide (NO^•) that subsequently activates endothelial and smooth muscle guanylate cyclase (G. cyclase). In the enzyme cycle, the trihydrobiopterin radical (BH₃^•) is generated, which is recycled by ascorbate (AA). The resulting ascorbate radical (AA^•−) is recycled by various NAD(P)H-dependent reductases. Failure to recycle BH₃^•, or its formation due to BH₄ oxidation by reactive oxygen species (ROS), results in the formation of dihydrobiopterin (BH₂), which competes with BH₄ for the enzyme. This, and loss of BH₄ uncouples eNOS, which then dissociates from the membrane into monomers (eNOS_m) that generate superoxide (O₂^•−) rather than NO^•. Reaction of O₂^•− with any available NO^• forms peroxynitrite, a strong nitrating oxidant. By initially recycling BH₄, ascorbate prevents loss of BH₄ and sustains eNOS activity. BH₄, tetrahydrobiopterin

FIG. 6.

Multiple mechanisms by which ascorbate preserves nitric oxide and tightens the endothelial permeability barrier. In endothelial cells in which NADPH oxidase (NOX) is activated by septic insult (or other mechanisms), the resulting superoxide (O₂^•−) reacts with available nitric oxide (NO^•) to form peroxynitrite (ONOO⁻), which nitrates and activates PP2A. The phosphatase then dephosphorylates occludin, causing it to pull away from the membrane and weaken tight junctional structures. Ascorbate prevents the activation of PP2A in this pathway by inhibiting NOX function and scavenging O₂^•− and ONOO⁻. In unstimulated cells (with presumably low levels of ONOO⁻, ascorbate also enhances nitric oxide generation by inhibiting PP2A by an unknown mechanism. This prevents PP2A from dephosphorylating and thus deactivating eNOS itself, as well as the AMP-dependent kinase (AMPK). The resulting increase in eNOS phosphorylation is mediated at least in part by phosphorylation-dependent activation of AMPK, which activates eNOS to generate nitric oxide. This, along with the preservation of BH₄ by ascorbate, increases intracellular nitric oxide, which then generates cyclic GMP through the canonical pathway to eventually tighten the endothelial permeability barrier. PP2A, protein phosphatase type 2A.

FIG. 7.

Role of ascorbate in maintaining the activity of α-ketoglutarate-dependent hydroxylation reactions. The proposed mechanism of α-ketoglutarate-dependent hydroxylases is first a sequential binding of ferrous iron, α-ketoglutarate, and molecular oxygen. At this point, there is the formation of a key ferryl-oxygen intermediate that not only usually binds the substrate protein (Pept), but can also generate an uncoupled cycle by cleaving α-ketoglutarate to succinate (Succ) and releasing CO₂. In the functional enzyme cycle, a proline or lysine is hydroxylated with subsequent sequential release of the modified protein, CO₂, and succinate leaving enzyme-bound ferrous iron that can restart the cycle. In an uncoupled cycle, the enzyme-bound iron is oxidized to ferric iron by oxygen, and the latter is released, perhaps as the hydroxyl radical (O^•−). The function of ascorbate is to reduce the enzyme-bound ferric to ferrous iron, thus allowing the enzyme to enter a potentially functional cycle again.