Thomas Schmitz-Rixen

Arteriogenesis versus angiogenesis: similarities and differences

Cardiovascular diseases account for more than half of total mortality before the age of 75 in industrialized countries. To develop therapies promoting the compensatory growth of blood vessels could be superior to palliative surgical surgical interventions. Therefore, much effort has been put into investigating underlying mechanisms. Depending on the initial trigger, growth of blood vessels in adult organisms proceeds via two major processes, angiogenesis and arteriogenesis. While angiogenesis is induced by hypoxia and results in new capillaries, arteriogenesis is induced by physical forces, most importantly fluid shear stress. Consequently, chronically elevated fluid shear stress was found to be the strongest trigger under experimental conditions. Arteriogenesis describes the remodelling of pre‐existing arterio‐arteriolar anastomoses to completely developed and functional arteries. In both growth processes, enlargement of vascular wall structures was proposed to be covered by proliferation of existing wall cells. Recently, increasing evidence emerges, implicating a pivotal role for circulating cells, above all blood monocytes, in vascular growth processes. Since it has been shown that monocytes/macrophage release a cocktail of chemokines, growth factors and proteases involved in vascular growth, their contribution seems to be of a paracrine fashion. A similar role is currently discussed for various populations of bone‐marrow derived stem cells and endothelial progenitors. In contrast, the initial hypothesis that these cells ‐after undergoing a (trans‐)differentiation‐ contribute by a structural integration into the growing vessel wall, is increasingly challenged.

The Range of Adaptation by Collateral Vessels After Femoral Artery Occlusion

Natural adaptation to femoral artery occlusion in animals by collateral artery growth restores only ≈35% of adenosine-recruitable maximal conductance (Cmax) probably because initially elevated fluid shear stress (FSS) quickly normalizes. We tested the hypothesis whether this deficit can be mended by artificially increasing FSS or whether anatomical restraints prevent complete restitution. We chronically increased FSS by draining the collateral flow directly into the venous system by a side-to-side anastomosis between the distal stump of the occluded femoral artery and the accompanying vein. After reclosure of the shunt collateral flow was measured at maximal vasodilatation. Cmax reached 100% already at day 7 and had, after 4 weeks, surpassed (2-fold) the Cmax of the normal vasculature before occlusion. Expression profiling showed upregulation of members of the Rho-pathway (RhoA, cofilin, focal adhesion kinase, vimentin) and the Rho-antagonist Fasudil markedly inhibited arteriogenesis. The activities of Ras and ERK-1,-2 were markedly increased in collateral vessels of the shunt experiment, and infusions of L-NAME and L-NNA strongly inhibited MAPK activity as well as shunt-induced arteriogenesis. Infusions of the peroxinitrite donor Sin-1 inhibited arteriogenesis. The radical scavengers urate, ebselen, SOD, and catalase had no effect. We conclude that increased FSS can overcome the anatomical restrictions of collateral arteries and is potentially able to completely restore maximal collateral conductance. Increased FSS activates the Ras-ERK-, the Rho-, and the NO- (but not the Akt-) pathway enabling collateral artery growth.

Elevated Fluid Shear Stress Enhances Postocclusive Collateral Artery Growth and Gene Expression in the Pig Hind Limb

Objective—The role of fluid shear stress (FSS) in collateral vessel growth remains disputed and prospective in vivo experiments to test its morphogenic power are rare. Therefore, we studied the influence of FSS on arteriogenesis in a new model with extremely high levels of collateral flow and FSS in pig and rabbit hind limbs. Methods and Results—A side-to-side anastomosis was created between the distal stump of one of the bilaterally occluded femoral arteries with the accompanying vein. This clamps the collateral reentry pressure at venous levels and increases collateral flow, which is directed to a large part into the venous system. This decreases circumferential wall stress and markedly increases FSS. One week after anastomosis, angiographic number and size of collaterals were significantly increased. Maximal collateral flow exceeded by 2.3-fold that obtained in the ligature-only hind limb. Capillary density increased in lower leg muscles. Immunohistochemistry revealed augmented proliferative activity of endothelial and smooth muscle cells. Intercellular adhesion molecule-1 and vascular cell adhesion molecule (VCAM)-1 were upregulated, and monocyte invasion was markedly increased. In 2-dimensional gels, actin-regulating cofilin1 and cofilin2, destrin, and transgelin2 showed the highest degree of differential regulation. Conclusions—High levels of FSS cause a strong arteriogenic response, reinstate cellular proliferation, stimulate cytoskeletal rearrangement, and normalize maximal conductance. FSS is the initiating molding force in arteriogenesis.

Immunosuppressive treatment of aortic allografts

Immunosuppression with cyclosporine (CsA) was explored as a means of preventing arterial allograft rejection and failure. Aortic allografts across the major histocompatibility barrier were studied in Brown-Norway and Lewis inbred rats. Grafts 1 cm long were interposed into the infra-abdominal aorta of Lewis recipients; five groups included two groups of untreated isograft and allograft control animals and three groups had allograft-CsA treatment regimens. The grafts were examined at 30, 60, and 100 days for patency, aneurysmal dilation, gross structural changes, inflammatory responses, and infiltration of W3/25- and OX8-positive lymphocytes. Only three allograft controls became occluded; the rest showed significant dilation (p less than 0.01), medial thinning and necrosis, intimal proliferation, and prominent cellular infiltration at 30 days. With all CsA regimens, aneurysmal dilation was significantly reduced or prevented (p less than 0.01), correlating with medial smooth muscle cell preservation. Cellular infiltration was delayed by an average daily dose of 5 to 10 mg/kg subcutaneous CsA for 30 days and was suppressed at 100 days by a continuous 5 mg/kg dose every 4 days. Intimal thickening in the graft was delayed but not prevented. We conclude that a low maintenance dose of CsA provides effective immunosuppression, thereby preventing aneurysm formation, and that the potential use of arterial allografts in vascular surgery may need to be readdressed.

Achieving the ideal properties for vascular bypass grafts using a tissue engineered approach: a review.

The multiple demands placed on small calibre cardiovascular bypass grafts have meant that a synthetic prosthesis with good long-term patency has not been developed. A tissue-engineered graft could fulfil the ideal characteristics present in an artery. However, the great disadvantage of such a conduit is the time necessary for maturation leading to unacceptable delays once the decision to intervene surgically has been made. This maturation process is essential to produce a graft which can withstand haemodynamic stress. Once implanted, the tissue-engineered graft can contract in response to immediate haemodynamic conditions and remodel in the long term. We review the latest tissue engineering approaches used to give the favourable properties of mechanical strength, arterial compliance, low thrombogenicity, long-term resistance towards biodegradation as well as technological advances which shorten the time required for production of an implantable graft.

Endovascular Treatment of Symptomatic Carotid Stenosis Using Stent Placement Long-Term Follow-Up of Patients With a Balanced Surgical Risk/Benefit Ratio

Background and Purpose— Carotid endarterectomy (CEA) is not necessarily beneficial in all patients with symptomatic high-grade (≥70%) internal carotid artery (ICA) stenosis. Independent risk factors modulate both the individual stroke risk under medical treatment and the combined stroke and death risk after CEA. Endovascular stenting of symptomatic ICA stenosis may be an alternative to CEA in patients with a balanced surgical risk/benefit ratio. Methods— We included 43 patients (71% men; median age, 67 years) with a recently symptomatic ICA stenosis with ≥70% luminal narrowing in whom the individual sum of medical and surgical risk factors suggested a balanced surgical risk/benefit ratio (risk-modeling appraisal derived from the European Carotid Surgery Trial). After stenting of the stenosed ICA with distal balloon protection, the mean±SD follow-up, including clinical and ultrasonographic examinations, was 20±11.8 months, with a median number of examinations of 5 per patient. Results— Recanalization of ICA stenoses was technically successful in 40 of 43 procedures (93%). Within the 30-day postinterventional period 1 death occurred (2.5%), and the combined stroke and death rate within follow-up was 5%. Except for 1 asymptomatic ICA occlusion, no restenosis ≥70% occurred during follow-up. Conclusions— ICA stenting in symptomatic patients with a balanced surgical risk/benefit ratio is technically feasible, with a low periprocedural risk of stroke or death. Furthermore, the risk of future stroke and rate of significant restenosis during long-term follow-up appears to be low, suggesting that ICA stenting may be useful in carotid revascularization and stroke prevention.

Trpv4 induces collateral vessel growth during regeneration of the arterial circulation

The development of a collateral circulation (arteriogenesis), bypassing an arterial occlusion, is important for tissue survival, but it remains functionally defective. Micro array data of growing collateral vessels, exposed to chronically elevated fluid shear stress (FSS), showed increased transcription of the transient receptor potential cation channel, subfamily V, member 4 (Trpv4). Thus, the aim of this study was to investigate the role of the shear stress sensitive Trpv4 in transmitting this physical stimulus into an active growth response. qRT‐PCR at different time points during the growth of collateral vessels after femoral artery ligature (FAL) in rats showed a strong positive correlation of Trpv4 transcription and the intensity of FSS. An increased protein expression of Trpv4 was localized in the FSS‐sensing endothelium by means of confocal immunohistochemistry. Cultured porcine endothelial cells showed a dose‐dependent expression of Trpv4 and an increased level of Ki67‐positive cells upon treatment with 4α‐Phorbol 12,13‐didecanoate (4αPDD), a specific Trpv4 activator. This was also demonstrated by flow culture experiments. These results were confirmed by in vivo application of 4αPDD in rabbit hind limb circulation via an osmotic mini‐pump after FAL. Trpv4 expression as well as Ki67‐positive staining was significantly increased in collateral vessels. Finally, 4αPDD treatment after FAL led to a 61% (215.5 ml/min/mmHg versus 350 ml/min/mmHg) recovery of conductance when compared with the non‐occluded artery. Cell culture and in vivo studies demonstrate that an FSS‐ or a 4αPDD‐induced activation of Trpv4 leads to an active proliferation of vascular cells and finally triggers collateral growth. Trpv4, a well‐known FSS‐sensitive vasodilator, has hitherto not been implicated in active growth processes of collateral arteries. This new function may lead to new therapeutic strategies for the treatment of arterial occlusive diseases.

Actin-Binding Rho Activating Protein (Abra) Is Essential for Fluid Shear Stress-Induced Arteriogenesis

Objective—Arteriogenesis, the development of a collateral circulation, is important for tissue survival but remains functionally defective because of early normalization of fluid shear stress (FSS). Using a surgical model of chronically elevated FSS we showed that rabbits exhibited normal blood flow reserve after femoral artery ligature (FAL). Inhibition of the Rho pathway by Fasudil completely blocked the beneficial effect of FSS. In a genome-wide gene profiling we identified actin-binding Rho activating protein (Abra), which was highly upregulated in growing collaterals. Methods and Results—qRT-PCR and Western blot confirmed highly increased FSS-dependent expression of Abra in growing collaterals. NO blockage by L-NAME abolished FSS-generated Abra expression as well as the whole arteriogenic process. Cell culture studies demonstrated an Abra-triggered proliferation of smooth muscle cells through a mechanism that requires Rho signaling. Local intracollateral adenoviral overexpression of Abra improved collateral conductance by 60% in rabbits compared to the natural response after FAL. In contrast, targeted deletion of Abra in CL57BL/6 mice led to impaired arteriogenesis. Conclusions—FSS-induced Abra expression during arteriogenesis is triggered by NO and leads to stimulation of collateral growth by smooth muscle cell proliferation.

The Role of Angiogenic Growth Factors in Arteriogenesis

Background/Aims: Collateral vessels restore only about 40% of the maximum dilatory reserve after femoral artery occlusion, whereas complete normalization is reached by increased fluid shear stress (FSS). We studied the role of known potent angiogenic growth factors (separately or in combination) in arteriogenesis by determining their expression in FSS-stimulated collaterals and close-to-collateral infusion of growth factor peptides in a rabbit model of femoral artery occlusion. Methods: Values of maximum collateral conductance (Cmax) and post mortem angiograms were compared to those achievable by high FSS. mRNA levels of growth factor ligands and receptors were determined in FSS-stimulated collaterals. Results: Seven days after vessel occlusion, FSS-stimulated legs showed a Cmax not significantly different from that of not occluded femoral arteries. Arteriogenesis was significantly less enhanced after growth factor treatment (MCP-1 86%, Ad5.1-FGF-4 75%, bFGF 72%, PDGF 64%, VEGF 50% of Cmax after FSS stimulation). RT-PCR showed no differential expression of FGF receptors, but an up-regulation of VEGF-receptor-2. Conclusion: The most potent known angiogenic growth factors at high pharmacological doses reach only a fraction of the maximum conductance obtained by high FSS. Arteriogenesis differs from angiogenesis, so the main focus to markedly improve arteriogenesis should be put on the underlying mechanisms of shear stress.

Effects of Endogenous Nitric Oxide and of DETA NONOate in Arteriogenesis

Previous studies showed that targeted endothelial nitric oxide synthase (eNOS) disruption in mice with femoral artery occlusion does not impede and transgenic eNOS overexpression does not stimulate collateral artery growth after femoral artery occlusion, suggesting that nitric oxide from eNOS does not play a role in arteriogenesis. However, pharmacologic nitric oxide synthase inhibition with L-NAME markedly blocks arteriogenesis, suggestive of an important role of nitric oxide. To solve the paradox, we studied targeted deletion of eNOS and of inducible nitric oxide synthase (iNOS) in mice and found that only iNOS knockout could partially inhibit arteriogenesis. However, the combination of eNOS knockout and treatment with the iNOS inhibitor L-NIL completely abolished arteriogenesis. mRNA transcription studies (reverse transcriptase-polymerase chain reaction) performed on collateral arteries of rats showed that eNOS and especially iNOS (but not neural nitric oxide synthase) become upregulated in shear stress-stimulated collateral vessels, which supports the hypothesis that nitric oxide is necessary for arteriogenesis but that iNOS plays an important part. This was strengthened by the observation that the nitric oxide donor DETA NONOate strongly stimulated collateral artery growth, activated perivascular monocytes, and increased proliferation markers. Shear stress-induced nitric oxide may activate the innate immune system and activate iNOS. In conclusion, arteriogenesis is completely dependent on the presence of nitric oxide, a large part of it coming from mononuclear cells.