Wei-Jun Cai

Arteriogenesis, a new concept of vascular adaptation in occlusive disease

The formation of collateral arteries as a process adaptive to arterial occlusion is now called ‘arteriogenesis’ to emphasize the difference to angiogenesis, the formation of capillaries by sprouting from pre-existent ones (W. Schaper, I. Buschmann. Cardiovasc Res 1999; 43: 835–7; I. Buschmann, W. Schaper. J Pathol 2000; 190: 338–42; D. Scholz et al. Virchows Arch 2000; 436: 257–70). The differences are that collaterals develop from pre-existing arterioles and that circulating monocytes adhere to endothelium that had been activated by the high shear stress generated by the large pressure differences between perfusion territories. Monocytes are the major producers of growth factors and of proteolytic enzymes that enable smooth muscle cells to migrate and divide. The nature of the growth factors remains uncertain. Neither FGF-1/2 nor VEGF is expressed on the transcriptional or translational level in collaterals proper and in the tissue surrounding them. Only FGF receptor 1 has a brief window of upregulation shortly after arterial occlusion. While transgenic overexpression of FGF-1 increases number and branching of arterioles, targeted disruption of FGF-1/2 does not negatively influence arteriogenesis. Cytokines that attract monocytes or prolong the life span of monocytes (MCP-1, GM CSF) are strong arteriogenic factors. Collateral vessels exhibit the same morphology whether they had formed in the heart, limbs or brain or in dogs, rabbits or mouse. They are tortuous because they also increase lengthwise in a restricted space. In animals larger than the mouse, they develop an intima, and initially, many arterioles participate in arteriogenesis, but only a few mature into large arterial channels which, when arterial occlusion had proceeded slowly enough, can replace the occluded artery to a significant proportion. Therapy with a single growth factor in animals with occluded femoral arteries significantly increases the speed of arteriogenesis but does not significantly increase the level of adaptation. It appears that the mastergene for arteriogenesis still awaits discovery.

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.

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.

Remodeling of the vascular tunica media is essential for development of collateral vessels in the canine heart.

Previous studies have shown that neointima formation and adventitial remodeling play an important role in the enlargement of collateral vessels (CVs) during coronary arteriogenesis in the dog heart. In this study, we investigated the importance of remodeling of the tunica media in the same model. Basal membrane (BM), contractile and cytoskeletal components of smooth muscle cells (SMCs) were studied in growth of coronary CVs induced by chronic occlusion of the left circumflex (LCX) coronary artery by routine histology, electron microscopy (EM), and immunoconfocal microscopy using antibodies against α-smooth actin (α-SM actin), calponin, desmin, and laminin. In addition, matrix metalloproteinase-2 (MMP-2) and tissue inhibitor-1 of matrix metalloproteinase (TIMP-1) were investigated. The data showed that (1) in normal small arteries (NVs) laminin formed a network in which SMCs were encaged;α-SM actin, calponin and desmin were evenly expressed in SMCs; (2) in early (2 weeks) growing CVs the laminin network was disrupted, desmin was significantly reduced in SMCs, but α-SM actin and calponin still highly expressed; (3) in actively (6 weeks) growing CVs laminin was still weak in the tunica media (TM), but without network-like structure. Desmin was further reduced in SMCs of TM, whereas α-SM actin and calponin showed little changes, although they were significantly decreased in intimal SMCs; (4) in mature CVs, the network-like structure was re-formed, and α-SM actin, calponin, and desmin were all similar to that in normal vessels; (5) histology for BM confirmed laminin staining; (6) EM revealed that in NVs the SMCs contained abundant contractile filaments and were surrounded by a layer of BM whereas in growing CVs, BM structure was not observed, but the SMCs in the media still contained many myofilaments; (7) MMP-2 was highly expressed in the media of early growing vessels, but decreased in TM of actively growing vessels where TIMP-1 expression was high. In conclusion, our data revealed features of TM of growing CVs. Disruption and degradation of BM facilitate SMC proliferation, and together with reduction of desmin and fragmentation of the internal elastic lamina enable the vascular wall to expand and enlarge when blood pressure and shear stress increase. MMP2 may be an important player in regulating SMC phenotype, proliferation, migration and maintaining integrity of the vascular wall through governing proteolysis during arteriogenesis. (Mol Cell Biochem 264: 201–210, 2004)

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.

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis.

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis. (Mol Cell Biochem 264: 193–200, 2004)

Comparative proteome analysis of 3T3-L1 adipocyte differentiation using iTRAQ-coupled 2D LC-MS/MS†

Adipose tissue is critical in obesity and type II diabetes. Blocking of adipocyte differentiation is one of the anti‐obesity strategies targeting on strong rise in fat storage and secretion of adipokine(s). However, the molecular basis of adipocyte differentiation and its regulation remains obscure. Therefore, we exposed 3T3‐L1 cell line to appropriate hormonal inducers as adipocyte differentiation model. Using iTRAQ‐coupled 2D LC‐MS/MS, a successfully exploited high‐throughput proteomic technology, we nearly quantitated 1,000 protein species and found 106 significantly altered proteins during adipocyte differentiation. The great majority of differentially expressed proteins were related to metabolism enzymes, structural molecules, and proteins involved in signal transduction. In addition to previously reported differentially expressed molecules, more than 20 altered proteins previously unknown to be involved with adipogenic process were firstly revealed (e.g., HEXB, DPP7, PTTG1IP, PRDX5, EPDR1, SPNB2, STEAP3, TPP1, etc.). The partially differential proteins were verified by Western blot and/or real‐time PCR analysis. Furthermore, the association of PCX and VDAC2, two altered proteins, with adipocyte conversion was analyzed using siRNA method, and the results showed that they could contribute considerably to adipogenesis. In conclusion, our data provide valuable information for further understanding of adipogenesis. J. Cell. Biochem. 112: 3002–3014, 2011.

Presence of Cx37 and lack of desmin in smooth muscle cells are early markers for arteriogenesis

In search of early structural markers of arteriogenesis, we studied the expression of gap junction proteins as well as of contractile and cytoskeletal proteins in smooth muscle cells (SMCs) during coronary collateral vessel growth induced by chronic occlusion of the left circumflex artery (LCx) in the dog heart. We used confocal microscopy with antibodies against connexin37 (Cx37), α-smooth muscle actin (α-SM actin), calponin, desmin and vinculin. The quantitative confocal analysis of immunofluorescence intensity showed that (1) in normal vessels (NV), Cx37 was present in endothelium only, not in SMC. Calponin, α-SM actin, desmin and vinculin were evenly expressed in SMC. (2) In early growing V (EV) with minimal intima formation, α-SM actin, calponin and vinculin showed little change in SMC, but desmin was 3.3 times lower than in NV, and Cx37 was induced (NV 0 arbitrary units/μm2, EV 50.3). (3) In actively growing V (AV), α-SM actin, calponin and vinculin were 3− , 3.3− and 2.9-fold lower, respectively, in the neointima as compared to the media. However, Cx37 was 48.2 AU/μm2 in the media and 15.8 AU/μm2in the neointima. Desmin was almost absent in the neointima and 5-fold reduced in the media. SMC, strongly positive for α-SM actin and calponin, expressed Cx37. Our findings indicate that induction of Cx37 and reduction of desmin precede the phenotypic changes of SMCs, which are characterized by down-regulation of α-SM actin, calponin and vinculin, and the formation of a neointima. An altered expression of Cx37 and desmin, therefore, are early markers for arteriogenesis in dog heart. (Mol Cell Biochem 262: 17–23, 2004)