Our data show that both endogenous NOS activity and exogenous NO modulate CAC motility. NOS activity is required for chemotactic migration of CACs to angiogenic chemokines, whereas exogenous NO induces chemokinesis, enhancing directional chemotaxis toward VEGF, without directly acting as a chemoattractant itself. Notably, the effects of the NO donor SNAP and NOS on the CACs were qualitatively similar to their effects on HUVECs, despite the presumption that these early pro-angiogenic CACs do not function as direct endothelial precursors. We show clinical relevance in that CAC migration in CAD patients is limited by decreased endogenous NOS activity due to impaired expression rather than impaired response to exogenous NO.
NOS plays an important regulatory role in vascular biology and defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction.27
An impairment of the endogenous NO signaling in endothelium is coupled to inability to produce an angiogenic response to VEGF.28,29
The effects of NO on CACs, which are important cells for endothelial repair, are not well understood. We describe here how the presence or absence of NO affects CAC motility. Corroborating previous studies,17,23,30
we demonstrate that CACs migrate to a number of chemokines NOS-dependently. This supports the notion that NOS represents an integral pathway for cell migration. We have recently shown that CACs migrate to a gradient of pleiotrophin (PTN) in a manner that is dependent on NOS, cGMP, NO, and PI3 Kinase.23
Similarly, it was previously shown by others that SDF-1α induces CAC migration in an eNOS, Akt, and PI3K dependent manner.30
We show here that the migration to VEGF involves the same pathways as migration to PTN and SDF-1α. This is important because a number of risk factors promoting arteriosclerosis and poor tissue regeneration, including smoking, aging, diabetes, hypertension, and hypercholesterolemia, have been shown to also inhibit NO production.2,17
These factors may mediate part of their vascular pathology by affecting vascular maintenance exerted by lowered NOS activity potentially via oxidative stress not only in endothelial cells, but also in CACs leading to dysfunction of these cells. This is supported by several papers in animal models and human clinical studies. In a recent clinical paper, we have shown that passive smoke may decrease CAC migration by blocking NO production.17
Animal hindlimb ischemia experiments have revealed that angiogenesis is impaired in eNOS −/− mice and that the eNOS substrate L-arginine can enhance angiogenesis in rabbits.27
Another study suggests that diabetes may impair re-endothelialization by impaired CAC function due to decreased eNOS expression.31
More recently, it was shown in diabetic rats and patients that diabetes may impair CAC functions by uncoupling eNOS.15
Taken together, our data suggest that dysfunctional CAC migration in CAD patients may be due to lower eNOS expression rather than impaired response to exogenous NO in CACs.
To our knowledge, this is the first paper to show the effect of exogenous NO on CAC migratory function. We show that an NO donor induces chemokinesis. It is important to note that this does not impair the CACs’ capacity to sense chemoattractant gradients and follow them, but actually significantly and additionally increases the net number of migrated cells at the site of higher chemokine concentration (). This is in agreement with previously published results by others showing that HUVECs cGMP-dependently migrate towards a gradient of NO using different NO-donors, DEA/NO and DETA/NO, which have a longer half life as compared to SNAP.32
To methodically exclude the possibility that SNAP merely increases cell proliferation at the lower side of the membrane, we performed proliferation assays showing that NO in fact decreases proliferation.33
Corroborating results by others, we show that SNAP also decreased apoptosis.34
In the context of the present study, we cannot exclude the possibility that NO increases survival of CACs and may thereby explain part of the migration results, potentially contributing to more cells recovered at the lower side of the membrane. Mechanistically, both CACs and HUVECs release NO and chemotaxis of both cell types is enhanced by NO-related chemokinesis. This suggests that NO may serve as a signal coordinating and potentially stimulating endothelial and pro-angiogenic cell interactions. NO and chemokines released by pro-angiogenic CACs that have homed to sites of injury may further attract new cells in a positive feedback loop via facilitating chemotaxis and chemokinesis. Once cells reach each other, higher NO levels may enhance adhesion (unpublished results Heiss et al.), and inhibit proliferation and apoptosis while facilitating even dispersal via chemokinesis of CACs and endothelial cells. As the observed effects were dose-dependent, the effect may likely differ between sites with different NO levels, such as inflammation with expression of high output iNOS (micromolar range) or vascular endothelium (low nanomolar range). Furthermore, these results may have clinical importance in disease states with lowered NO bioavailability, e.g. decreased levels of plasma S-nitrosothiols or nitrite which represent physiologic ‘NO donors’ with cardiovascular risk factors.2,24,35,36
Schematic to illustrate the proposed additive effects of directional (chemotaxis) and non-directional random cell movement (chemokinesis).
Our data further support the concept that the NOS/NO pathway is a strong modulator of CAC functions as it is in endothelial cells. CAC functions are likely to be affected both by factors that impair this pathway of endothelial cells in patients with cardiovascular disease in vivo
and by reduced NO bioavailability.2